Chromosome 3p21.3 genes are tumor suppressors

ABSTRACT

Tumor suppressor genes play a major role in the pathogenesis of human lung cancer and other cancers. Cytogenetic and allelotyping studies of fresh tumor and tumor-derived cell lines showed that cytogenetic changes and allele loss on the short arm of chromosome 3 (3p) are most frequently involved in about 90% of small cell lung cancers and greater than 50% of non-small cell lung cancers. A group of recessive oncogenes, Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), Luca 1 (HYAL1), Luca 2 (HYAL2), PL6, 123F2 (RaSSFI), SEM A3 and Beta* (BLU), as defined by homozygous deletions in lung cancers, have been located and isolated at 3p21.3.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/217,112, filed Jul, 10, 2000.

[0002] The U.S. Government has rights in the invention by virtue ofP50-CA70907.

BACKGROUND OF THE INVENTION

[0003] I. Field of the Invention

[0004] The invention generally relates to the fields of molecularbiology and oncology.

[0005] II. Related Art

[0006] Cancer is the result in the occurrence of multiple factors.Mutations may occur in proto-oncogenes that cause cellular proliferationto increase. Mutations also may occur in tumor suppressors whose normalfunction is to regulate cellular proliferation. Mutations in DNA repairenzymes impair the ability of the cell to repair damage beforeproliferating. Tumor suppressor genes are normal genes whose absence(loss or inactivation) can lead to cancer. Tumor suppressor genes encodeproteins that slow cell growth and division. Cancer arises when there isa mutation in both alleles.

[0007] Tumor suppressor genes (TSGs) play a major role in thepathogenesis of human lung cancer and other cancers. Lung cancer cellsharbor mutations and deletions in multiple known dominant and recessiveoncogenes^(6,7). Known TSGs such as Rb, p53, and putative TSGs have beenfound at chromosome regions 3p, 5q, 6p, 8p, 9p, and 11p as well as othersites^(6,8,9). Cytogenetic and allelotyping studies of fresh lung tumorsand tumor cells showed tumor-cell allele loss at multiple sites,suggesting the existence of one or more such TSGs^(6-8,10). However,cytogenetic changes and allele loss on the short arm of chromosome 3(3p) have been shown to be most frequently involved in about 90% ofsmall cell lung cancers (SCLCs) and >50% of non-small cell lung cancers(NSCLCs)^(6,8,10,11). SCLC and NSCLC are the two treatment groups oflung tumors and are made up of four histological types. Squamous cell-,adeno-, and large cell carcinomas are in the NSCLC group. Small celllung cancer is in the SCLC group. Approximately 75% of lung tumors areNSCLCs. Metastases occur later with NSCLC than with SCLC. SCLC is one ofthe most metastatic of solid tumors⁵². In addition, similar 3p changeshave been seen in several other cancers in addition to lung, such asrenal^(12,13), breast^(14,15), head and neck¹⁶, pancreatic¹⁷, kidney¹⁸,oral¹⁹, and uterine cervical cancers^(20,21). Furthermore, a group ofTSGs, as defined by homozygous deletions in lung cancers, have beenlocated and isolated at 3p21.3 in a 450-kb region^(6,10,22-24). Studiesof lung cancer preneoplasia indicate that 3p21 allele loss is theearliest genetic abnormality in lung cancer detected so far, occurringin hyperplastic lesions; this shows that one or more 3p-recessiveoncogenes function as “gatekeepers” in the molecular pathogenesis ofmany human cancers, including lung cancer, where it is likely to beinvolved in >50% of all cases^(6,10,22-26).

[0008] Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines;candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). The evidenceshows that genes in this 3p21 critical region are involved in regulationof the telomerase-mediated cellular immortality pathway in lung, renal,and breast cancer cells^(28,29). It has also been shown that 3p deletionoccurs more frequently in the lung tumor tissues of patients who smoke.In addition, elevated sensitivity to the carcinogen benzo[a]pyrene diolepoxide at 3p21.3 has been associated with an increased risk of lungcancer, suggesting that 3p21.3 is a molecular target of carcinogens inlung cancer³¹. Despite those studies, there remains a need to furtheridentify the functions of these genes and demonstrate their involvementwith cancer.

SUMMARY OF THE INVENTION

[0009] The tumor suppressor genes at 3p21.3 are now disclosed: Gene 26(CACNA2D2)³⁴⁰, PL6, Beta* (BLU), LUCA-1 (HYAL1l), LUCA-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), and SEM A3. The function of theindividual 3p genes in suppression of tumor growth and tumorprogression, induction of apoptosis, alteration of cell cycle kinetics,and repression of telomerase activity has been characterized by theliposome- and recombinant adenoviral vector-mediated transfer of 3pgenes in vitro and in vivo. This also is the initial disclosure of theBeta* gene.

[0010] Therefore, it is an objective of the present invention to providemethods of using tumor suppressors having a chromosomal location of3p21.3. It is also an objective to provide a tumor suppressor, Beta*.Further, it is an objective to provide methods of constructingrecombinant adenovirus in which these tumor suppressors may be inserted.

[0011] An embodiment of the present invention is an isolatedpolynucleotide encoding a polypeptide comprising an amino acid sequenceof SEQ ID NO:2. There is also provided a nucleic acid with the sequenceof SEQ ID NO: 1. Further provided is an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:2. Another embodiment is a nucleicacid of 15 to about 100 base pairs comprising from 15 contiguous basepairs of SEQ ID NO:1, or the complement thereof. A further embodimentincludes from about 20, 25, 30, 40, 50 or 100 contiguous base pairs ofSEQ ID NO: 1, or the complement thereof.

[0012] Another embodiment of the invention is an isolated peptide havingbetween 10 and about 50 consecutive residues of SEQ ID NO:2. Further,the peptide may comprise 15, 20, 25, or 30 consecutive residues of SEQID NO:2. In this application, “about” is defined as within + or −2 aminoacids.

[0013] Yet another embodiment is an expression cassette comprising apolynucleotide encoding a polypeptide having the sequence of SEQ IDNO:2, wherein said polynucleotide is under the control of a promoteroperable in eukaryotic cells. In another embodiment, the promoter ofthis expression cassette is heterologous to the coding sequence. Thepromoter may be a tissue specific and inducible promoter. In anotherembodiment, the expression cassette may be contained in a viral vector.The viral vector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, or a herpesviralvector. In a further embodiment the expression cassette may comprise apolyadenylation signal.

[0014] Another embodiment is a cell comprising an expression cassettecomprising a polynucleotide encoding a polypeptide having the sequenceof SEQ ID NO:2, wherein said polynucleotide is under the control of apromoter operable in eukaryotic cells, said promoter being heterologousto said polynucleotide.

[0015] Yet another embodiment of the invention is a monoclonal antibodythat binds immunologically to a polypeptide comprising SEQ ID NO:2, oran immunologic fragment thereof. Also provided is a monoclonal antibodywith a detectable label. The label may be a fluorescent label, achemiluminescent label, a radiolabel and an enzyme. Another embodimentof the invention is a hybridoma cell that produces a monoclonal antibodythat binds immunologically to a polypeptide comprising SEQ ID NO:2, oran immunologic fragment thereof. A further embodiment is a polyclonalantisera, antibodies of which bind immunologically to a polypeptidecomprising SEQ ID NO:2, or an immunologic fragment thereof.

[0016] Yet another embodiment is a isolated and purified nucleic acidthat hybridizes, under high stringency conditions, to a DNA segmentcomprising SEQ ID NO: 1, or the complement thereof. In a furtherembodiment the nucleic acid is about 15, 17, 20 or 25 bases in length.

[0017] Another embodiment of the invention is a method for constructinga recombinant adenovirus comprising: (a) providing a shuttle vector,said shuttle vector comprising an adenoviral inverted terminal repeat(ITR) sequence, an expression cassette comprising a promoter and apoly-A sequence, a transgene under the control of said promoter, andunique restriction sites at the 5′- and 3′-ends of theITR-promoter-transgene-poly-A segment; (b) cutting at said restrictionenzyme sites; (c) ligating the released segment into an adenoviralvector lacking the entire E1 and E3 regions and transforming theresulting vector a bacterial host cell; (d) obtaining vector from saidbacterial host cell and digesting the vector to release theE1/E3-deleted adenovirus genome; and (e) transfecting the adenovirusgenome into E1-expressing host cells. In a further embodiment, thetransgene is Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.In another embodiment, the promoter may be a cytomegalovirus (CMV)promoter and said poly A sequence is bovine growth hormone (BGH) poly Asequence.

[0018] Yet another embodiment of the invention is a method forconstructing a recombinant adenovirus comprising: (a) providing ashuttle vector comprising an adenoviral inverted terminal repeat (ITR)sequence, an expression cassette comprising a promoter and poly-A signalsequence, a transgene under the control of said promoter, a tetracyclineresistance-off responsive element, and unique restriction sites at the5′ and 3′ ends of the IRT-promoter-transgene-poly-A segment; (b) cuttingat said restriction enzyme sites; (c) ligating the released segment intoan adenoviral vector comprising a tetracyclin resistant-offtransactivator gene and lacking the entire E1 and E3 regions, andtransforming the resulting vector a bacterial host cell; (d) obtainingvector from said bacterial host cell and digesting the vector to releasethe E1/E3-deleted adenovirus genome; and (e) transfecting the adenovirusgenome into E1-expressing host cells. In a further embodiment, thetransgene is Gene 26, PL6, Beta*, LUCA-1, LUCA-2, 123F2, Fus1, 101F6,Gene 21 or SEM A3. In another embodiment, the promoter may be acytomegalovirus (CMV) promoter and said poly A sequence is bovine growthhormone (BGH) poly A sequence.

[0019] In yet another embodiment, also provided is a shuttle vectorcomprising an adenoviral inverted terminal repeat (ITR) sequence, anexpression cassette comprising a promoter and poly-A sequence, aTetR-Off responsive element, and unique restriction sites at the 5′- and3′-ends of the ITR-promoter-poly-A segment. In another embodiment of theinvention the promoter is a cytomegalovirus (CMV) promoter and said polyA sequence is bovine growth hormone (BGH) poly A sequence. Also providedis a multipurpose cloning site in said segment, positioned between saidpromoter and said poly-A sequence.

[0020] Yet another embodiment is an adenoviral vector comprising atetracycline resistant-off transactivator gene and lacking the entire E1and E3-regions.

[0021] Another embodiment of the invention is a method of diagnosingcancer in a subject comprising the steps of: (i) obtaining a biologicalsample from said subject; and (ii) assessing the expression of afunctional Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2(HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 productin sample. In a further embodiment the sample is a tissue sample. Thetissue sample may be brain, lung, liver, spleen, kidney, lymph node,small intestine, blood cells, pancreas, colon, stomach, cervix, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,oral tissue, bone marrow or blood tissue. In another embodiment, theassessing comprises detecting a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3. Detecting maycomprise amplification said nucleic acid, nucleic acid hybridization, orsequencing. In another embodiment, assessing comprises detecting a Gene26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide. Thedetecting of a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide maycomprise ELISA or immunohistochemistry. In yet another embodiment, theassessing may comprise wild-type or mutant oligonucleotidehybridization, with said oligonucleotide configured in an array on achip or wafer. In another embodiment of the invention, the expression ofGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 is compared withthe expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3in normal samples. In another embodiment, the comparison involvesevaluating the level of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),SEM A3 expression.

[0022] Another embodiment is a non-human transgenic animal lacking oneor both functional alleles of Gene 26 (CACNA2D2), PL6, Beta* (BLU),Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21(NPRL2), SEM A3. Also provided is a non-human transgenic animal thatoverexpresses Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 as compared to asimilar non-transgenic animal. In a further emodiment is a non-humantransgenic animal, the genome of which comprises an expression cassettecomprising a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 under the control ofan inducible promoter.

[0023] An embodiment of the invention is a method for suppressing growthof a tumor cell comprising contacting said cell with an expressioncassette comprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2),PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1,101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3; and (b) a promoter active in said tumor cell, underconditions permitting the uptake of said nucleic acid by said tumorcell. In another embodiment, the tumor cell is derived from a braintumor, lung tumor, liver tumor, spleen tumor, kidney tumor, lymph nodetumor, small intestine tumor, blood cell tumor, pancreatic tumor, colontumor, stomach tumor, cervix tumor, breast tumor, endometrial tumor,prostate tumor, testicle tumor, ovarian tumor, skin tumor, head and necktumor, esophageal tumor, oral tissue tumor, or bone marrow tumor. In afurther embodiment, the nucleic acid is contained in a viral vector. Theviral vector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. In yet another embodiment, the nucleic acid iscontained in a liposome.

[0024] Another embodiment of the invention is a method of altering thephenotype of a tumor cell comprising contacting said cell with anexpression cassette comprising: (a) a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 10F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), SEM A3; and (b) a promoter active in said tumor cell, underconditions permitting the uptake of said nucleic acid by said tumorcell. In another embodiment, the phenotype is selected from the groupconsisting of proliferation, migration, contact inhibition, soft agargrowth, cell cycling, invasiveness, tumorigenesis, and metastaticpotential. In yet another embodiment, the promoter is a cytomegalovirus(CMV) promoter.

[0025] Another embodiment is a method of inhibiting cancer in a subjectsuffering therefrom comprising administering to said subject anexpression cassette comprising: (a) a nucleic acid encoding Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3 polypeptide; and (b) a promoter active in tumorcells of said subject, whereby expression of said polypeptide inhibitssaid cancer. In a further embodiment, the subject is a human. In otherembodiments, the nucleic acid encodes Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3. In another embodiment, the cancer is a selected from thegroup consisting of brain cancer, lung cancer, liver cancer, spleencancer, kidney cancer, lymph node cancer, small intestine cancer, bloodcell cancer, pancreatic cancer, colon cancer, stomach cancer, cervixcancer, breast cancer, endometrial cancer, prostate cancer, testiclecancer, ovarian cancer, skin cancer, head and neck cancer, esophagealcancer, oral tissue cancer, and bone marrow cancer. In yet anotherembodiment, the expression cassette is contained in a viral vector. Theviral vector may be a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector. In another embodiment, the expression cassette iscontained in a lipsome. In another embodiment, the expression cassettefurther comprises a poly-A sequence. The poly-A sequence may be a bovinegrowth hormone (BGH) poly-A sequence. In a further embodiment, theexpression cassette is administered intratumorally, in the tumorvasculature, local to the tumor, regional to the tumor, or systemically.

[0026] Also provided in the method of inhibiting cancer is theadministering of a chemotherapuetic agent to said subject. In anotherembodiment, the chemotherapeutic comprises cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, gemcitabien, navelbine, farnesyl-proteintansferase inhibitors, transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate. Also provided is the administeringradiation to said subject. In another embodiment, the radiation isdelivered local to a cancer site or is whole body radiation. Theradiation may comprise γ-rays, X-rays, accelerated protons, microwaveradiation, UV radiation or the directed delivery of radioisotopes totumor cells. In yet another embodiment, a a second anticancer gene maybe administered to said subject. The second anticancer gene may be atumor suppressor. The second anticancer gene may be an inhibitor ofapoptosis. In another embodiment, the second anticancer gene is anoncogene antisense construct.

[0027] An embodiment of the invention is a method of treating a subjectwith cancer, comprising the step of administering to said subject a Gene26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 polypeptide. In anotherembodiment, the cancer is a selected from the group consisting of braincancer, lung cancer, liver cancer, spleen cancer, kidney cancer, lymphnode cancer, small intestine cancer, blood cell cancer, pancreaticcancer, colon cancer, stomach cancer, cervix cancer, breast cancer,endometrial cancer, prostate cancer, testicle cancer, ovarian cancer,skin cancer, head and neck cancer, esophageal cancer, oral tissuecancer, and bone marrow cancer. In a further embodiment, the polypeptideis contained within a liposome. the liposome may be comprised ofN-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP) andcholesterol. In another embodiment, the subject is human.

[0028] Another embodiment of the invention is a method of screening acandidate substance for anti-tumor activity comprising the steps of: (i)providing a cell lacking a functional Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3 polypeptide; (ii) contacting said cell with saidcandidate substance; and (iii) determining the effect of said candidatesubstance on said cell. In another embodiment, the cell is a tumor cell.In another embodiment, the determining may comprises comparing one ormore characteristics of the cell in the presence of said candidatesubstance with the same one or more characteristics of a similar cell inthe absence of said candidate substance. In a further embodiment, thecharacteristic is selected from the group consisting of Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 expression, phosphataseactivity, proliferation, metastasis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression,metastasis and tissue invasion. In another embodiment, the candidatesubstance is a chemotherapeutic or radiotherapeutic agent. Also providedis a candidate substance selected from a small molecule library. Infurther embodiments, the cell is contacted in vitro or in vivo.

[0029] An embodiment of the invention is a method of screening acandidate substance for anti-tumor activity comprising the steps of: (i)providing a cell; (ii) contacting said cell with said candidatesubstance; and (iii) determining the effect of said candidate substanceon expression of a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3polypeptide.

[0030] Another embodiment is a method of producing a Beta* polypeptidein a host cell comprising: (a) providing an expression cassettecomprising a nucleic acid encoding Beta* operably linked to an promoteractive in said host cell; (b) transferring said expression cassette intosaid host cell; and (c) culturing said host cell under conditionspermitting expression of said Beta* polypeptide.

[0031] Yet another embodiment of the invention is a method of diagnosingcancer in a subject comprising the steps of: (i) obtaining a biologicalsample from said subject; and (ii) detecting hypermethylation of thepromoter region of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.

[0032] Other objects, features and advantages of the present inventionwill become apparent from the following detailed description. It shouldbe understood, however, that the detailed description and the specificexamples, while indicationg preferred embodiments o the invention, aregiven by way of illustration only, since various changes andmodificaitons within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF SUMMARY OF THE DRAWINGS

[0033] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

[0034]FIG. 1. Scheme of construction and production of recombinantadenovirus using pAd-RAP and pAd-RAP-Shuttle system.

[0035]FIG. 2. Scheme of construction of recombinant adenovirus usingpAd-RAP-Tet-Off and pAd-RAP-TRE-CMV-Shuttle. TetR-Off=tetracyclinresistant-off transactivator gene, TRE=TetR-Off responsive elements.

[0036]FIG. 3. Timing of genetic changes found in preneoplastic lesionsof the respiratory epithelium associated with primary non-small celllung cancers.

[0037]FIG. 4. Allelotyping of 3p region in DNAs from human lung cancercell lines and tumors. Filled ovals=loss of heterozygosity; openovals=retaining of alleles; and hatched ovals=homozygous deletions.

[0038]FIG. 5. Scheme of the location of the 3p21 tumor suppressor regionin human chromosome 3p and the structure of recombinant adenoviralvectors of 3p genes. The sizes of the individual 3p genes and theircorresponding amino acid residues, and the active tumor suppressor (TS)regions and known TSGs in the 3p are also indicated.

[0039]FIG. 6. Effects of overexpression of 3p genes on tumor cell growthin Ad-3p-transduced lung cancer cells and normal human bronchialepithelial cells. MOIs were expressed as viral particles/cell (vp/c).

[0040]FIG. 7. Quantification of adenovirus-mediated 3p gene expressionin H1299 cells by Real Time RT-PCR. The MOIs are expressed as viralparticles/cell (vp/c).

[0041]FIG. 8. Induction of apoptosis by overexpression of 3p genes inAd-3p-transduced lung cancer cells and normal HBEC. Apoptosis wasanalyzed by FACS with TUNEL reaction.

[0042]FIG. 9. Effect of overexpression of 3p genes on cell cyclekinetics in Ad-3p-transduced human lung cancer cells A549 and H1299.

[0043]FIG. 10. Effect of overexpression of 3p genes on A549 tumor growthby intratumoral injection of Ad-3p vectors in nude mice.

[0044]FIG. 11. Effect of overexpression of 3p genes on A549 lungmetastatic tumor growth by systemic injectionof protamine-Ad-3p vectorcomplexes in nude mice.

[0045]FIG. 12. Map of the RASSF1 locus, transcripts, and proteindomains, A) The exon—intron structure of the RASSF1 locus with thelocation of the CpG islands in the predicted promoter regions (thelocations of which are shown by double-headed arrows) of RASSF1A andRASSF1C. RASSF1A transcription is predicted to come from the mostcentromeric promoter region located within a CpG island and begins withexon 1A. RASSEiF also commences at this promoter but is missing exon iC.Transcription of RASSFIC is predicted to begin in the most telornericpromoter region, which is approximately 2 kilobases from that of RASSF1Aand begins with exon 1. Blocks represent exons; lines represent introns.B) Schematic of the RASSF1A transcript and predicted protein-sequencedomains. The location of the various primers (PKCDF, NF, R182, and R292)used for isoform-specific reverse transcription (RT)-polymerase chainreaction (PCR) analyses are indicated. Tick marks identify the exonboundaries. The potential arc homology 3 (5H3)-binding region, putativediacylglycerol (DAG)-binding domain, PEST sequence, Rasassociationdomain, and ataxia-telangiectasia-mutated (ATM) phosphorylation site arelabeled. C) Schematic of the RASSFIC transcript and predictedprotein-sequence domains. The locations of the various primers (NOX3,R182, and R292) used for isoform-specific RT-PCR analyses are indicated.D) Schematic of the RASSFIF transcript and predicted protein-sequencedomains.

[0046]FIG. 13. RASSF1A and RASSF1C messenger RNA levels detected byisoform-specific reverse transcription-polymerase chain reaction(RT-PCR) in a sampling of lung cancer cell lines (A), breast cancerlines (B), and resected lung tumors and normal human lung and breastepithelial cultures (C). All RT-PCR products were separated on 2%agarose gels and were identified by staining with ethidium bromide.Arrows indicate location of transcripts. A) Lung cancer lines tested inlanes: 1-11157; 2=11358; 3=11727; 4=11740; 5=11748; 6=11838; 7=111184;8=111299; 9=111304; 10=111437; 11=111450; 12=111770; 13=111792;14=111963; 15 111993; 16=112009; 17=112077; iS =112108; 19=11HCC44; and20=HCC78. B) Breast cancer lines tested in lanes:1=11CC38;2=11CC1187;3=HTB19;4=HTB2O;5=HTB22; 6=11TB23; 7=11TB24;8=11TB25; 9=11TB26; 10=11TB27; 11=HTB12I; 12=HTB129; 13 HTB13O;14=HTBI31; 15=HTB132; 16=H‘I’B133; 17=11CC 1395; iS=11CC 1428;19=11CC1569; 20=11CC1806; and 21=11CC2157. C) Resected lungadenocarcinoma samples (ADC 1-5) and cultures of normal small-airwayepithelial cells (SAECs), normal human bronchial epithelial (NHBE)cultures, and normal human breast epithelial (NHBRE) cultures.

[0047]FIG. 14. Expression of RASSF1A after treatment of lung cancercells with 5-aza-2′-deoxycytidine (SAza-CdR). NCI-11157, anon-small-cell lung carcinoma (NSCLC) cell line that expresses RASSF1Cbut not RASSFIA, was grown in the presence (+lanes) and absence (−lanes)of 0.5 p.M SAza-CdR for 48 hours. Total RNA was isolated, complementaryDNA was prepared, and isoformspecific reverse transcription-polymerasechain reaction was performed for RASSF1A, RASSF1C, andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control.

[0048]FIG. 15. Methylation-specific polymerase chain reaction (PCR) forthe detection of methylated RASSF1A 5, CpG sequences in primary resectednon-small-cell lung carcinomas (NSCLCs) and their accompanying normallung tissue (upper panel), small-cell lung carcinoma (SCLC) cell lines(middle panel), and primary breast cancers (lower panel). Representativesamples are shown. For resected NSCLCs, U=results with primers specificfor unmethylated sequences; M=results with primers specific formethylated sequences. NL=normal lung tissue; T=tumor; P=results withperipheral blood lymphocyte DNA, which is unmethylated or in vitromethylated (IVMD); and 1120=negative controls with water blanks. ForSCLCs, each lane shows the PCR results for the methylated sequences froma different cell line. Lane 20 is negative control. For the breastcancers, each lane shows the PCR results for methylated sequences from adifferent sample. PCR products were separated on 2% agarose gels andbands were detected after staining with ethidium bromide.

[0049]FIG. 16. Kaplan-Meier survival curve for 107 patients withresected non-small-cell lung carcinomas based on RASSF1A methylationstatus (32 methylated and 75 not methylated), For the patients withunmethylated RASSF1A alleles, the number of cases=75, censored=39, andevents=36, with a mean overall survival of 52 months (95% confidenceinterval [CI] =44 to 59) and a median overall survival of 49 months (95%CI=44 to 59); for the patients with methylated RASSF1A alleles, thenumber of cases=32, censored=nine, and events=23, with a mean overallsurvival of 37 months (95% CI=27 to 46) and a median overall survival of28 months (95% CI=9 to 47). The log-rank test statistic for equality ofsurvival distributions for RASSF1A methylation was 3.97, with df 1,P=0.0463. The patients at risk for each group were: RASSF1Aunmethylated-12 months (n=63), 36 months (n=34), and 60 months (n=16);RASSF1A methylated-12 months (n=24), 36 months (n=13), and 60 months(n=5).

[0050]FIG. 17. Effect of RASSF1A on the in vitro and in vivo growth ofthe non-small-cell lung carcinoma (NSCLC) cell line NCI-111299. A)Anchorage-dependent and anchorage-independent colony formation aftertransfection of NCI-H1299 cells with the˜ioo empty vector (pcDNA3.1+) orpeDNA3.1+ expression vectors containing wild-type p53 or RASSF1A. Foranalysis of anchorage-dependent growth, after 2 days in nonselectivegrowth medium, transfected NCI-111299 cells were diluted into 100-mm²dishes with selective medium. Transfected cells were plated in liquidmedium (for anchorage-dependent assays) or soft agar (foranchorage-independent assays) containing 800 p.g/mL of G418. Colonieswere stained with methylene blue in anchorage-dependent experimentsafter 14 days. Results represent the average of eight to 12 experimentsin liquid medium and three soft-agarexperiments. Standard deviations areshown or are less than 2%. Solid bars=anchorage-dependent growth (95%confidence interval [CI]=0 to 36 for wt-p53 (wild-type) and 52 to 60 forRASSFIA); open bars=anchorage-independent growth (95% CI=0 to 6 forwild-type (wt)-p53 and 0 to 39 for RASSFIA). B) Northern blot analysisof the RASSF1A expression in stable clones of NCI-H1299 cellstransfected with the pcDNA3.1+ vector or pcDNA3.1+ containing RASSF1Acomplementary DNA (cDNA). The vector control (vector) and four separateclones with various RASSF1A messenger RNA levels are shown. Several ofthese clones were used in the anchorage-independent growth assay shownin D. Ethidium bromide staining of the ribosomal RNA is shown as aloading control. The clones were also verified to express the RASSF1Aisoform by reverse transcription—polymerase chain reaction with the useof isoform-specific primers. C) Soft-agar (anchorage-independent) colonyformation in stable clones of NCI-111299 cells transfected with thepcDNA3.1+ vector or pcDNA3.1+ containing RASSF1A cDNA. The means andstandard deviations are shown. For each of the RASSFIA expressingclones, the 95% CI=0 to 4 for F1A.4, 2 to 16 for F1A.5, and 3 to 14 forF1A.19. D) NCI111299 cells were infected with the pBABEpuro retrovirusexpression vectors containing either the vector control or the RASSF1Aor RASSF1C cDNAs. Infected cells (10000 per plate) were suspended in0.33% agar, and the suspension was layered over a 0.5% agar base.Colonies greater than 0.2 mm in diameter were counted after 21 days. Thelower right panel shows a representative western blot, developed with arabbit antibody to the RASSF1-glutathione S-transferase fusion protein,to verify the expression of the RASSF1 proteins. C=positive controlgenerated by transient transfection of NCI-111299 cells with peDNA3.1+containing RASSF1A cDNA; V=infection of NCI-H1299 cells with theretroviral vector control (note runover from positive control;1A=infection of NCI-H1299 cells with the retroviral vector containingRASSF1A; and 1C=infection of NCI-H 1299 cells with the retroviral vectorcontaining RASSF1C. E) Effect of RASSF1A on the in vivo growth ofNCI-111299 cells. Approximately 10⁷ viable NCI-H 1299 cells expressingRASSF1A were injected into the flanks of each of five previouslyirradiated BALB/c (nulnu) nude mice. Tumor size was monitored overtime,and size is shown in cubic millimeters. The average volume of tumorsgrown in more than 20 mice that were given an injection ofvector-transfected NCI-H 1299 cells is shown (H1299 parent). Mice thatwere given an injection of RASSFIA-infected NCI-H 1299 cells grew nomeasurable tumors.

[0051]FIG. 18. Schematic representation of the location of the putative3p21.3 tumor suppressor region in human chromosome 3p and the structureof the recombinant adenoviral vectors of 3p21.3 genes. The sizes of theindividual 3p21.3 genes and their corresponding amino acid residuesdeduced from coding sequences of cDNAs, and the active tumor suppressor(TS) regions and known TSGs in the 3p are indicated. The recombinantadenoviral vectors of 3p21.3 genes (Ad-3ps) were constructed byinserting a mammalian expression cassette in which the 3p21.3 gene wasdriven by a CMV promoter and tailed with BGH poly A signal sequence intothe E1-deleted region of the replication incompetent adenovirus type 5(Ad5) genome. The relative locations of E1-deletion (ΔE1) andE3-deletion (ΔE3), the inverted repeated terminal (IRT) sequences in theAd5 genome are indicated.

[0052]FIG. 19. Effects of exogenous expression of 3p21.3 genes on tumorcell growth in Ad-3p-transduced human lung cancer cells and normalbronchial epithelial cells. Cells were transduced with adenoviralvectors of 3p21.3 genes, 101F6, NPRL2, BLU, RASSF 1C FUS1, HYAL2, andHYAL1, control genes, LacZ and p53, and empty vector, Ad-EV, at highestMOIs (vp/c), 5000 for A549, 1000 for H1299, 5000 for H460, 2500 forH358, and 1000 for HBE, respectively, and PBS alone was used as a mockcontrol. The cell viability was expressed as the percentage of viableadenoviral vector-transduced cells in relation to PBS-treated controlcells (100%). The error bars represent standard deviations of the meanin at least three individual experiments. Treatments were given inquadruplicate for each experiment. The significance of the difference incell viability between vector-treated cells and the Ad-EV-, Ad-LacZ-, orPBS-treated controls was analyzed by two-sided Student's T-test. P<0.05was taken as significant. The differences between the cell viability ofthe Ad-EV- and Ad-LacZ-transduced cells versus PBS-treated controls werenot significant (P=0.25 to P=0.95 from different time points and celllines). The differences between the cell viability of the Ad-101F6,Ad-Fus1, and Ad-NPRL2- transduced cells versus the Ad-EV-,Ad-LacZ-transduced, or PBS-treated controls at same MOIs weresignificant in A549, H1299, and in H460 at both 3 days and 5 daysposttransduction. (P≦0.0001 to P≦0.005) but not significant in H358 andHBEC cell lines at both 3 and 5 days posttransduction (P≧0.10 to P≧0.95,from different time points and cell lines), respectively. The effects ofAd-BLU, Ad-HYAL2, and Ad-HYAL1 on cell viability were not significant inall cell lines (P>0.45) compare to those of Ad-EV and Ad-LacZ.

[0053]FIG. 20. Quantification of adenovirus-mediated 3p21.3 geneexpression in H1299 cells by real-time RT-PCR. The real-time RT-PCR wasperformed and PCR profiles were generated by an ABI Prism 7700 SequenceDetection system and equipped software (Perkin Elmer AppliedBiosystems). Known concentrations of β-Actin DNA were used as astandard. The H1299 cells were transduced by adenoviral vectors of3p21.3 genes, FUS1 (A), 101F6 (B), NPRL2 (C), and HYAL1 (D) at a MOI of1, 5, and 10 pfu/cell for 48 hr, respectively, as indicated by arrows.

[0054]FIG. 21. Induction of apoptosis by exogenous expression of 3p21.3genes in Ad-3p-transduced human NSCLC cells and normal HBECs. Apoptosiswere analyzed by FACS, using TUNEL reaction with FITC-labeled dUTP.Cells were transduced with adenoviral vectors of 3p21.3 genes at an MOIs(vp/c) of 5000 for A549 (A), 1000 for H1299 (B), 5000 for H460 (C), 2500for H358 (D), and 1000 for HBEC (E), respectively, and PBS, Ad-EV, andp53 were used as controls. Cell were harvested and analyzed forapoptosis at the indicated days posttransduction. The rate of apoptosisis expressed as the percentage of FITC-labeled cells in the total cellpopulation. The error bars represent standard deviations of the mean intwo or three repeated experiments with triplicate treatments and TUNELreactions for each experiment. The significance of the difference inapoptosis between vector-treated cells and the Ad-EV-, Ad-LacZ-, orPBS-treated controls was analyzed by two-sided Student's T-test. P<0.05was considered significant. The differences between the apoptosisinduced by the Ad-EV- and Ad-LacZ-transduced cells versus PBS-treatedcontrols were not significant (P=0.925 to P=0.675 from different timepoints and cell lines). The differences between the apoptosis induced inthe Ad-101F6, Ad-FUS1, and Ad-NPRL2-transduced cells versus the Ad-EV-,Ad-LacZ, or PBS-treated controls were significant in A549 and H460 cellsat both 3 days and 5 days posttransduction (P≦0.0001 to P≦0.005), andsignificant versus the Ad-EV- and PBS-treated cells in H1299 at 5 daysposttransduction (P≦0.02), but not significant in H358 and HBEC celllines at both 3 and 5 days posttransduction at all time points (P≧0.85to P≧0.95), respectively. Induction of apoptosis in Ad-p53-transducedH358 cells were significant at all time points compared to all othertreatments (P<0.0001). Induction of apoptosis in cells treated withAd-BLU, Ad-HYAL2, and Ad-HYALyal1 was not significant compared to thosetreated with PBS, Ad-EV, or Ad-LacZ, in all cell lines at all timepoints (P>0.85).

[0055]FIG. 22. Effects of intratumoral administration of adenoviralvectors of 3p21.3 genes on growth of human lung cancer A549 (A) andH1299 (B) subcutaneous tumors in nu/nu mice. When the tumor reached 5 to10 mm in diameter at about 2 weeks after tumor inoculation, the tumorwas injected with individual adenoviral vectors of 3p21.3 genes, 101F6,NPRL2, BLU, RASSF1CFUS1, HAYL2, and HYAL1 or control vectors Ad-EV,LacZ, and p53, at a dose of 5×10¹⁰ vp/tumor each in 200 μl of PBS forthree times within a week, respectively, and PBS alone was used as amock control. Results were reported as the mean ±SD in 5-10 mice foreach treatment group. Tumor volumes were normalized by the percentageincrease of tumor sizes after treatment relative to those at thebeginning of the treatment in each group. Mean tumor volumes±SE fromthese experiments are shown. ANOVA was performed to determinestatistical significance between each treatment group using a Statisticasoftware (StatSoft Inc.) and P≦0.05 was considered significant. Thedifferences betweof en the tumor volumes ofin the Ad-101F6, Ad-FUS1,Ad-NPRL2 -treated mice versus in the Ad-EV- and Ad-LacZ- treated mousecontrols were statistically significant in both A549 and H1299 tumormodels (P<0.0001), and the difference in the Ad-HYAL2-treated mice wassignificant in A549 (P=0.024) but not in H1299 tumor models, after 5days from the last injection (P<0.0001), but not significant inAd-HYAL1, Ad-HYAL2, Ad-RASSF1C, and Ad-BLU-treated (P>0.05 in both A549and H1299 tumor models).

[0056]FIG. 23. Effect of systemic administration of protamine-Ad-3pcomplexes on development of A549 experimental lung metastases in nu/numice. A., Relative metastatic tumors in mice treated with P-Ad-3p21.3genes. All animals were i.v. injected with various protamine-adenoviralvector complexes every other two (lays for 3 times each at a dose of3×10¹⁰ viral particles plus 300 μg protamine in a total volume of 200 μlper animal, and PBS alone was used as a mock control. Each treatmentgroup consisted of 5-10 animals. Lungs were harvested two weeks afterthe last injection and metastastic colonies on the surfaces of lung werecounted without knowledge of the treatment groups. Development ofmetastases were represented as the percentages of metastatic coloniesformed in protamine-adenovirus complexes-treated groups in relation tothose in the PBS-treated group (as 100%). Error bars represent asstandard error (SE). Non-parametric t-test (Wald-Wolfowitz Runs Test)was performed to determine statistical significance between eachtreatment group using a Statistica software (StatSoft Inc.) and P≦0.05was considered significant. A significant inhibition of development ofmetastases was observed in mice treated with P-Ad-101F6 (P=0.002),P-Ad-NPRL2 (P=0.001), P-Ad-BLU (P=0.018), P-Ad-FUS1 (P=0.002), andP-Ad-HYAL2 (P=0.014), respectively, compared to mice treated with PBS,P-Ad-EV, or P-Ad-LacZ, but no significant inhibition in mice treatedwith. P-Ad-BLU (P=0.818) or P-Ad-HYAL1 (P=0.904). B., the representativephotos of lungs stained with India ink for metastases. The metastaticcolonies were shown as white spots on the surfaces of lung.

[0057]FIG. 24. (a) RT-PCR Analysis of NSCLCs cDNA HCC515 (Wild typeFUS1) and H322 (smaller cDNA mutant form of FUS1). (b) Genomic structureof wild type FUS1 and the mutant aberrant slicing form. Top line isgenomic DNA from cosmid clone LUCA#13 (#Z84492) and the indicatednucleotide sequence numbers. Arrowheads indicated primers for SSCPanalysis. Boxes represent cDNA with the open reading frames (black) anduntranslated regions (white) for the 110 amino acid wild type and 82amino acid aberrant splice form of FUS1. Note the sequence for FUS1 andFUS1-aberrant is the same for the first 80 amino acids. Three sets ofprimers were designed to cover the full FUS1 open reading frame forPCR-SSCP analysis. The primers used were S1: GTTATGGTAGTGCGGACTG andAS1, GGTGGAACCATTGCCCTTAC; S2. GACCTGTGACATTTGCCGTG and AS2,CAACAGATCCCATCTGGGTC: S3; and CCTGAGCTGACCCCTTACA and AS3,TCTGTCTGCCACCTCCCAG.

[0058]FIG. 25. (a.) Western blot analysis of endogenous and transientexpression of FUS1 in lung cancer cells. Transfection was performedaccording to the manufacture's instruction using DMRIE C (LifeTechnologies, Inc., GIBCO BRL Gaithersburg, Md.). NSCLC H1299 (2×10⁵cells) were plated in 3.5 cm dishes 24 hour before transfection and 2 μgof plasmid and 4 μl of DMRIE C were used for each transfection. All ofthe plasmids were resequenced after PCR construction and the sequencesof the various FUS1 open reading frames were verified. Ten μl of lysatewas made from 2×10 ⁴ cells using sample buffer (100 mM Tris 2% SDS 10%β-mercaptoethanol 20% glycerol 0.03% PBP) and run in 12.5% SDS-PAGE gelsfollowed by transfer to nitrocellulose membranes. After blocking with 5%dry milk and 0.2% Tween 20 in PBS, the membranes were incubated at roomtemperature for 1 h with rabbit polyclonal antibodies. Anti FUS1antibodies (1:300 dilution of sera) were generated by immunizing rabbits(Strategic Biosolution Ramona, Calif.) with peptides corresponding toamino acid 1 to 15 of the human FUS1 protein sequence. Anti-FLAGantibody M2 was from Sigma (St. Louis, Mo.). The membranes weredeveloped after incubation with presence of peroxidase-labeledanti-rabbit or anti-mouse IgG antibodies using Super Signalchemiluminescent substrate (Pierce Rockford, Ill.). The calculatedmolecular weight of FLAG-tagged FUS1 is 15 kd and the size of the bandthat was recognized by both antibodies is slightly higher than thecalculated size. As expected the mutant FUS1 (predicted to be 82 aminoacids) is slightly smaller than wild type FUS1 (110 amino acids). (b.)Results of colony formation assays in H1299 NSCLC cells. Aftertransfection, the H1299 cells were trypsinized, replated and cultured inG418 (600 μg/ml) supplemented medium (RPMI 1640 5% fetal bovine serum )for 2 or 3 weeks and the number of G418 resistant colonies counted afterstaining with methylene blue in ethanol/PBS (50/50%). Note dramaticsuppression of colony formation after transfection with FUS1 andFUS1-FLAG but much less suppression with the 82 amino acid aberrant FUS1construct. The mean and standard deviations for an average of 2-4 platesfor 2 or more experiments for H1299 were: vector control pcDNA3.1,100±18% (100%=248 colonies), FUS1-FLAG 16±10%, FUS1 23±11%, FUS1 mutant77±11%. Colony numbers of FUS1 and FUS1-FLAG transfected cells weresignificantly reduced (P<0.01, student's t test) compared with vectorcontrol. H322 cells had 40±34% colony formation with FUS1-FLAGtransfection compared to 100% for vector control (P<0.05).

[0059]FIG. 26. (a.) Induction of FUS1 protein by Ecdysone expressionvector (Invitrogen, Carlsbad, Calif.) Under the control of thePonasterone A in NCI-H1299 stable transfected clones. The inventorstransfected the regulatable hormone receptor vector pVgRXR into H1299and obtained 20 Zeocin (selection marker of pVgRXR) resistant clones.These stable pVgRXR transfectants were screened for β-gal activityfollowing transfection with pIND-LacZ. From these clones the inventorsselected clone ECR 9 as a parent cell line in which β-gal activity wasspecifically regulated by Ponasterone A in H1299 cells. The inventorsmade an expression vector which contained FUS1-FLAG (pIND sp1-FUS1-FLAG) and transfected this into ECR 9. Western analysis. Ten μgtotal cell lysate protein from each cell line and anti-FUS1 antibodywere used for the analysis. The concentration (μM) of Ponasterone A usedfor induction is indicated above the blots. ECR9 is H1299 parent cellline transfected with the regulatory vector alone; clones 13 and 16represent H1299 clones containing a regulatable FUS1 vector. The invitro growth of (b.) NSCLC H1299ECR 9 (control), (c.) H1299FUS1Clone13and (d.) H1299FUS1Clone16 was measured by the MTT assay. Cells (10⁴)were plated in 1 ml of RPMI 1640 (Life Technologies Inc.) with 5% fetalbovine serum and cultured in the presence (1, 5 μM) or absence ofPonasterone A in a 24 well plates (added at day 0) and wells wereharvested for MTT assays at the days indicated. MTT (Sigma) was added tothe cultures (500 μg/ml), incubated at 37° C. for 2 hours, theintracellular formazan crystals solubilized with isopropanol containing0.01N HCl, and the absorbance of the solution at 560 nm was measuredusing a spectrophotometer. The OD 560 is directly proportioned to cellnumber in the range of 0-1.2. Data points represent an average of 3wells with SD (contained within the symbols) of each data point ˜5%. Forcell cycle distribution analysis of the FUS1 inducible H1299 clones,cells (2×10⁵) of ERC 9, CL.13 and Cl. 16 were plated on 10 cm dishes andcultured in the presence (5 μM) or absence of Ponasterone A for 2 days.Cells were harvested, fixed in 50% ethanol/PBS, treated with 5 mg/mlRNase, stained with propidium iodide and analyzed for DNA content byFACSCaliber instrument (Becton Dickinson San Jose, Calif.). FACSanalysis was performed in three independent experiments with similarresults. Under FUS1 induced conditions the % of cells in G1 increasessignificantly (P<0.05) compared to controls.

SEQUENCE SUMMARY

[0060] SEQ ID NO: 1=Beta* (BLU) nucleotide sequence

[0061] SEQ ID NO: 2=Beta* (BLU) amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

[0062] Tumor suppressor genes (TSGs) play a major role in thepathogenesis of human lung cancer and other cancers. Lung cancer cellsharbor mutations and deletions in multiple known dominant and recessiveoncogenes^(6,7). Other TSGs that have been found to be altered in lungcancer are p53, p16, Rb, and FHIT-1⁵². Known TSGs such as Rb, p53, andothers have been found at chromosome regions 3p, 5q, 6p, 8p, 9p, and 11pas well as other sites^(6,8,9). Cytogenetic and allelotyping studies offresh lung tumors and tumor cells showed tumor-cell allele loss atmultiple sites, suggesting the existence of one or more suchTSGs^(6-8,10). These loci are important in understanding predispositionto lung cancer among smokers⁵². Loss of heterozygosity (LOH) is commonin lung cancers, as in other solid tumors. Some of the chromosomal locithat experience a loss of heterozygosity in lung cancer are: 9p21-p22,13q14, 17p13.1, 3p12-p14, 3p21, 3p25, 5q21, 11q12-q24, and 22q.Vulnerability to lung cancer may be due to genetic differences occurringat multiple loci. These genes may play a role in the metabolization oftobacco carcinogens. Cytogenetic changes and allele loss on the shortarm of chromosome 3 (3p) have been shown to be most frequently involvedin about 90% of small cell lung cancers (SCLCs) and >50% of non-smallcell lung cancers (NSCLCs)^(6,8,10,11). In addition, similar 3p changeshave been seen in several other cancers, such as renal^(12,13),breast^(14,15), head and neck¹⁶, pancreatic¹⁷, kidney¹⁸, oral¹⁹, anduterine cervical cancers^(20,21).

[0063] Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines.Candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). The evidenceshows that genes in this 3p21 critical region are involved in regulationof the telomerase-mediated cellular immortality pathway in lung, renal,and breast cancer cells^(28,29). Cell hybrid and microcell chromosome 3transfer studies have demonstrated the ability of human chromosome 3genes to suppress malignancy in human lung, renal, and ovarian cancercell lines^(6,30). It also has been shown that 3p deletion occurs morefrequently in the lung tumor tissues of patients who smoke. In addition,elevated sensitivity to the carcinogen benzo[a]pyrene diol epoxide at3p21.3 has been associated with an increased risk of lung cancer,suggesting that 3p21.3 can be a molecular target of carcinogens in lungcancer³¹.

[0064] This invention identifies genetic loci involved in lung cancer. Agroup of TSGs (Fus1, 101F6, Gene2l (NPRL2), Gene26 (CACNA2D2), PL6,Luca1 (HYAL1), Luca2 (HYAL2), 123F2 (RASSF1), Beta* (BLU) and SEM A3),as defined by homozygous deletions in lung cancers, have been locatedand isolated at 3p21.3 in a 450-kb region^(6,10,22-24). Studies of lungcancer preneoplasia indicate that 3p21 allele loss is the earliestgenetic abnormality in lung cancer detected so far. One or more3p-recessive oncogenes function as “gatekeepers” in the molecularpathogenesis of many human cancers, including lung cancer, where it islikely to be involved in >50% of all cases^(6,10,22-26) (FIG. 3).

[0065] Since (1) the 3p genes located at 3p21.3 in a 450 kb region aredefined by homozygous deletions in lung cancers; (2) the 3p21 alleleloss is one of the earliest genetic abnormalities detected in lungcancer and other tumors; (3) the loss of heterozygosity, the homozygousdeletion, and the abnormality of these 3p genes are associated with thepathogenesis of many human cancers including lung cancer where it islikely to be involved in >50% of all cases; and (4) the multiple 3pgenes function as tumor suppressor genes or the 3p21.3 region as a tumorsuppressor region, the technologies and molecular tools developed basedon the genetic/cytogenetic status and function of these 3p genes areextremely valuable for the early detection, diagnosis, and monitoring ofprevention and therapeutic efforts for various human cancers.

[0066] I. Function of 3p Genes as Tumor Suppressor Gene Region

[0067] One of the criteria for defining the role of genes as tumorsuppressor genes is to demonstrate that the tumor phenotype marked byinactivation of the genes can be rescued by the replacement of thewild-type alleles of these genes. If the frequent loss of heterozygosity(LOH), homozygous deletion, or, in some cases, abnormal transcripts andmutations of genes are the targets of carcinogens and the loss offunction of genes leads to human cancers, then replacement of theabnormal genes with the wild-type genes would result in tumorsuppression similar to that shown by the Rb or p53 tumor suppressor geneincluding inhibition of tumor cell growth in vitro, suppression oftumorigenicity and tumor growth, and inhibition of tumor cell invasionand metastasis in vivo³²⁻³⁴.

[0068] The identification of the 3p genes as tumor suppressor genes wasbased on the cytogenetic and alleotyping studies of fresh tumors andtumor cell lines showing tumor cell allele loss at multiple sites andhomozygous deletion in this region. Some of these 3p genes share varieddegrees of homology in DNA and the predicted amino acid sequences tosome known genes in the presently available data bases; however, thefunction of these 3p genes or the 3p21.3 region in pathogenesis andtumorigenesis of cancers is previously unknown. Cell hybrid andmicrocell chromosome 3 transfer studies demonstrated the ability ofhuman chromosome 3 genes to suppress malignancy in human lung, renal,and ovarian cancer cell lines and mouse A9 fibrosarcoma cells, however,only one example involving introduction of a whole chromosome 3 intoA549 human lung carcinoma cells has been reported^(10,30,36-38).

[0069] In the present invention, it is the first time that the functionof the individual 3p genes in suppression of tumor growth and tumorprogression, induction of apoptosis, alteration of cell cycle kinetics,as well as repression of telomerase activity has been characterized bythe liposome- and the recombinant adenoviral vector-mediated transfer of3p genes in vitro and in vivo, and that the concept of function of 3pgenes as a tumor suppressor region has been developed based on the tumorsuppressor activities involved in multiple 3p genes in this critical3p21.3 region. The finding of the 3p tumor suppressors permits newtherapeutics to be developed for treating related cancers.

[0070] The adenoviral vector has been shown to be the most efficientgene delivery system in vitro and in vivo^(4,5). Recombinant adenovirusvectors have been widely used for gene transfer in basic research aswell as for clinical applications¹⁻³. However, in vitro manipulation ofadenoviral DNA is very difficult due to the large size of the genome andlimited unique and useful restriction sites, making the construction ofrecombinant adenoviral vectors relatively time consuming and laborintensive. Two conventional methods for the construction suchrecombinant adenoviruses are well documented: an in vitro ligationmethod³⁹ and an in vivo homologous recombination method⁴⁰. The in vitroligation method consists of a first step of subcloning the transgeneinto a plasmid vector to generate a segment containing the left end ofthe viral genome and a mammalian gene expression cassette, and then therecombinant vector is produced by in vitro ligation of the segment intothe viral genome, followed by transfection of the reconstitutedrecombinant viral molecule into permissive 293 cells. Hiroyuki and Kaydisclose an in vitro ligation method⁴⁵. The other methods use twoplasmids with overlaping fragments to generate the recombinant virus byhomologous recombination in 293 cells. The major limitations for thesemethods are the generation of a background of nonrecombinant virus, lowfrequency of in vivo homologous recombination, and repeated screening ofplaque to isolate pure recombinant vectors. There are severalalternative procedures for construction of recombinant adenoviralvectors based on homologous recombination of the two plasmidscotransfected in 293 cells⁴⁰, the targeted modification of theadenoviral genome in an infectious yeast artificial chromosome (YAC) inyeast cells⁴¹, the cosmid adenoviral vectors in cosmid packagingbacteria⁴², and plasmids in recA⁺ bacteria strain^(43,44). These methodswhile more efficient, are more complex, require the use of an additionalyeast hosts or nonconventional bacterial strain, face the low frequencyof homologous recombination in these host and the instability of therecombinant adenoviral genome in plasmids hosted by the recA⁺ bacterialstrain.

[0071] By comparison, the present Ad-RAP system is very simple,efficient, and rapid for the construction of recombinant adenoviralvector for gene therapy. This system requires a simple in vitro ligationusing regular molecular biology reagents and commonly used bacterialstrain. The resulting recombinant adenoviral genome containing plasmidscan be easily screened and are stable. The subsquent transfection of thelinearized recombinant adenovirus DNA mediated by liposome (DOTAP) intothe permissive 293 cells is very efficient and a homogeneous populationof recombinant adenovirus can be produced rapidly.

[0072] The recombinant adenoviral vector, Ad-3ps, can be used to deliver3p genes in vitro and in vivo with a much higher efficiency than anyother available gene delivery systems and technologies. Due to the highefficiency of transduction and high level expression of transgenes invarious cell types mediated by adenoviral vectors, the Ad-3p vectors canbe used as a effective tool to study the biological function andmechanisms of these tumor suppressor genes in vitro and in vivo. TheAd-3ps can be used to limit tumorigenicity, tumor suppression, andrestriction of metastatic processes in various tumors such as lung,colon, breast, stomach, cervix, and head and neck, prostate, andpancreas by either intravenous or intratumoral injection of the Ad-3pvector or protamine-Ad-3p complexes.

[0073] In many cases, expression of some genes such as Bak, Bax, FasLare highly toxic to the host 293 cells, making construction andproduction of the recombinant adenovirus bearing such genes extremelydifficult and some times impossible by any of the above methods andprocedures. The present Ad-RAP-TetR-Off system can be used tosuccessfully construct and produce such recombinant adenoviral vectors.The expression of the transgene in the adenoviral vector can be turnedoff by addition of tetracycline into the cell culture medium, and,consequently, the toxic effect of the gene on the host cells can beavoided and the recombinant adenovirus can be produced in the 293 cellsas usual. Some other systems such as binary adenoviral vector systems⁴⁶have been developed to successfully construct such recombinant adenovralvectors. However, the expression of a transgene in one viral vectordepends on the expression of a trans-activator gene in another one,i.e., two adenoviral vectors are required for transgene expression invitro and in vivo, which, in turn, limited the application of such asystem in vivo. By comparision, in the Ad-TetR-Off vector system, thetrans-activator TetR-Off gene and the TetR-Off response element (TRE)co-exist in the same adenoviral vector, and, therefore, expression oftransgene can be turned on or off in one vector in the absence orpresence of the tetracycline inducer. Furthermore, since the transgeneis under the control of the TRE regulatory promoter, the level ofexpression of the transgene can be efficiently regulated byadministration of tetracycline in vitro and in vivo. Together, thesenovel features of the Ad-RAP-Tet-Off system make it a useful new toolfor rapid and successful construction and production of a recombinantadenoviral vector caryring cytotoxic genes.

[0074] Introduction of individual wild-type 3p21.3 genes by liposome-and adenovirus-mediated transient transfection into lung cancer celllines containing either heterozygous or homozygous deletion of the 3pregion inhibited tumor cell growth, induced apoptosis, and altered cellcycle kinetics, suppressed tumor growth and tumor progression in nudemice. Varied levels of inhibition of cell growth, induction ofapoptosis, and alteration of cell cycle kinetics were observed inAd-Fus1, Ad101F6, and Ad-Gene 21-transduced human lung cancer cellsH1299, A549, and H460, which are either lacking in 3p genes or haveabnormal ones. However, no significant inhibitory effects on cell growthwere observed in Ad-Fus1, Ad-101F6, and Ad-Gene 21-transduced normalHBEC and H358 cells, which contain wild type 3p genes. Therefore, theobserved cell growth inhibition was not due to the general cytotoxicityof these genes. The overexpression of 3p genes in these Ad-3ptranfectants was verified by a quantitative Real-Time RT-PCR. Tumorgrowth was significantly suppressed by overexpression of 101F6, Fus1,and Gene 21 via intratumoral injection of Ad-101F6, Ad-Fus1, and Ad-Gene21 vectors in H1299 and A549 xenografts in nude mice. Furthermore, thelung metastatic tumor growth was also significantly inhibited bysystematic injection of protamine-complexed Ad-101F6, Ad-Fus1, andAd-Gene 21 in nude mice bearing the experimental A549 metastasis.Together, these results show that multiple 3p genes function as tumorsuppressor genes or as a tumor suppressor region in vitro and in vivo,and that these newly identified and characterized 3p tumor suppressorgenes or this 3p tumor suppressor region can be used for cancer genetherapy, using molecular tools such as the liposome-3p complexes,recombinant adenoviral vectors containing 3p genes, and the local orsystematic gene delivery systems developed in this invention. Theidentification and functional characterization of the wild-type 3p21.3genes and their mutated forms in lung cancer and other cancers providesa crucial step in the development of therapy for lung cancer and othertumors

[0075] A. Background of 3p21.3

[0076] A group of TSGs, as defined by homozygous deletions in lungcancers, have been located and isolated at 3p21.3 in a 450-kbregion^(6,10,22-24). Studies of lung cancer preneoplasia indicate that3p21 allele loss is the earliest genetic abnormality in lung cancerdetected so far, occurring in hyperplastic lesions. One or more3p-recessive oncogenes function as “gatekeepers” in the molecularpathogenesis of many human cancers, including lung cancer, where it islikely to be involved in >50% of all cases^(6,10,22-26).

[0077] Recently, human chromosome band 3p21.3 has been shown to undergooverlapping homozygous deletions in several SCLC and NSCLC lines.Candidates of TSGs have been located in this critical region in severalhuman cancers, further defining a TSG region^(6,10,24,27). Genes in the3p21 critical region are involved in regulation of thetelomerase-mediated cellular immortality pathway in lung, renal, andbreast cancer cells^(28,29). It has also been shown that 3p deletionoccurs more frequently in the lung tumor tissues of patients who smoke.In addition, elevated sensitivity to the carcinogen benzo[a]pyrene diolepoxide at 3p21.3 has been associated with an increased risk of lungcancer, suggesting that 3p21.3 can be a molecular target of carcinogensin lung cancer³¹.

[0078] B. 3p21.3 Proteins

[0079] In addition to the entire Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3 molecules, the present inventionalso relates to fragments of the polypeptides that may or may not retainthe tumor suppressing activity. The entire length of each protein isFus1=161, 101F6=222, Gene 21=203, Gene 26=1205, Beta*=440, Luca1=435,Luca2=473, PL6=351, 123F2=431, and SEM A3=749 amino acids. Fragments,including the N-terminus of the molecule may be generated by geneticengineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 molecules withproteolytic enzymes, known as proteases, can produce a variety ofN-terminal, C-terminal and internal fragments. Examples of fragments mayinclude contiguous residues of the Beta* sequence of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,50, 55, 60, 65, 75, 80, 85, 90, 95, 100, or more amino acids in length.These fragments may be purified according to known methods, such asprecipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

[0080] 1. Purification of 3p21.3 Proteins

[0081] It may be desirable to purify Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; sodium dodecyl sulfate/polyacrylamide gelelectrophoresis (SDS/PAGE); isoelectric focusing. A particularlyefficient method of purifying peptides is fast protein liquidchromatography (FPLC) or even HPLC.

[0082] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis.

[0083] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0084] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE (Capaldiet al., 1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

[0085] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample can be low because the bands are sonarrow that there is very little dilution of the sample.

[0086] Gel chromatography, or molecular sieve chromatography, is aspecial type of partition chromatography that is based on molecularsize. The theory behind gel chromatography is that the column, which isprepared with tiny particles of an inert substance that contain smallpores, separates larger molecules from smaller molecules as they passthrough or around the pores, depending on their size. As long as thematerial of which the particles are made does not adsorb the molecules,the sole factor determining rate of flow is the size. Hence, moleculesare eluted from the column in decreasing size, so long as the shape isrelatively constant. Gel chromatography is unsurpassed for separatingmolecules of different size because separation is independent of allother factors such as pH, ionic strength, temperature, etc. There alsois virtually no adsorption, less zone spreading and the elution volumeis related in a simple matter to molecular weight.

[0087] Affinity Chromatography is a chromatographic procedure thatrelies on the specific affinity between a substance to be isolated and amolecule that it can specifically bind to. This is a receptor-ligandtype interaction. The column material is synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial is then able to specifically adsorb the substance from thesolution. Elution occurs by changing the conditions to those in whichbinding will not occur (alter pH, ionic strength, temperature, etc.).

[0088] The matrix should be a substance that itself does not adsorbmolecules to any significant extent and that has a broad range ofchemical, physical and thermal stability. The ligand should be coupledin such a way as to not affect its binding properties. The ligand shouldalso provide relatively tight binding. It should be possible to elutethe substance without destroying the sample or the ligand. One of themost common forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

[0089] The present invention also describes smaller Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3-relatedpeptides for use in various embodiments of the present invention.Because of their relatively small size, the peptides of the inventionalso can be synthesized in solution or on a solid support in accordancewith conventional techniques. Various automatic synthesizers arecommercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young, (1984); Tam et al.,(1983); Merrifield, (1986); and Barany and Merrifield (1979), eachincorporated herein by reference. Short peptide sequences, or librariesof overlapping peptides, usually from about 6 up to about 35 to 50 aminoacids, which correspond to the selected regions described herein, can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides. Alternatively, recombinant DNA technologymay be employed wherein a nucleotide sequence which encodes a peptide ofthe invention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

[0090] The present invention also provides for the use of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 proteinsor peptides as antigens for the immunization of animals relating to theproduction of antibodies. A biospecific or multivalent composition orvaccine is produced. It is envisioned that the methods used in thepreparation of these compositions will be familiar to those of skill inthe art and should be suitable for administration to animals, i.e.,pharmaceutically acceptable.

[0091]2. Variants of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, and SEMA3

[0092] Amino acid sequence variants of these polypeptides can besubstitutional, insertional or deletion variants. Deletion variants lackone or more residues of the native protein that are not essential forfunction or immunogenic activity. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additionsare called fusion proteins.

[0093] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of this kind preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or plienylalanine; and valine toisoleucine or leucine.

[0094] The following is a discussion based upon changing of the aminoacids of a protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

[0095] Amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine, and isoleucine.

[0096] C. Nucleic Acids

[0097] Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,and SEM A3 are found at a chromosomal position of 3p21.3 in a 450 kbcritical region. They are found in the following order at 3p21.3: Gene26, PL6, 101F6, Gene 21, Beta*, 123F2, Fus1, Luca2, Luca1, and SEM A3.The length of each is Fus1=1696, 101F6=1117, Gene 21=1696, Gene 26=5482,Beta*=1746, Luca1=2565, Luca2=1783, PL6=1860, 123F2=1502, and SEMA3=2919 nucleic acids(FIG. 5).

[0098] In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, “Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 genes” may contain a variety of different bases and yet stillproduce a corresponding polypeptide that is functionallyindistinguishable, and in some cases structurally, genes disclosedherein.

[0099] Nucleic acids according to the present invention may encode anentire Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,and SEM A3 genes, a domain of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3, or any other fragment of the Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3sequences set forth herein. The nucleic acid may be derived from genomicDNA, i.e., cloned directly from the genome of a particular organism. Inother embodiments, however, the nucleic acid would comprisecomplementary DNA (cDNA).

[0100] The term “cDNA” is intended to refer to DNA prepared usingmessenger RNA (mRNA) as template. The advantage of using a cDNA, asopposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There may be times whenthe full or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

[0101] It also is contemplated that a given Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 from a given species maybe represented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (Table 1).

[0102] As used in this application, the term “polynucleotide having thenucleic acid sequence of SEQ ID NO: 1 ” refers to a nucleic acidmolecule that has been isolated free of total cellular nucleic acid. Afunctionally equivalent codon is a codon that encodes the same aminoacid, such as the six codons for arginine or serine (Table 1), and alsorefers to codons that encode biologically equivalent amino acids. TABLE1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGCUGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0103] The DNA segments of the present invention include those encodingbiologically functional equivalent Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, and SEM A3 proteins and peptides, as describedabove. Such sequences may arise as a consequence of codon redundancy andamino acid functional equivalency that are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

[0104] D. Hybridization

[0105] Naturally, the present invention also encompasses DNA segmentsthat are complementary, or essentially complementary, to the sequencesencoding Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2and SEM A3. Nucleic acid sequences that are “complementary” are thosethat are capable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary” meansnucleic acid sequences that are substantially complementary, as may beassessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to the aforementioned nucleicacid segment under relatively stringent conditions such as thosedescribed herein. Such sequences may encode the entire Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 protein orfunctional or non-functional fragments thereof.

[0106] Alternatively, the hybridizing segments may be shorteroligonucleotides. Sequences of 17 bases long should occur only once inthe human genome and, therefore, suffice to specify a unique targetsequence. Although shorter oligomers are easier to make and increase invivo accessibility, numerous other factors are involved in determiningthe specificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, or 1000 bases and longer arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots, in situ tissue hybridizationand as primers in amplification reactions.

[0107] Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNAs and/or RNAs or to provide primers foramplification of DNA or RNA from samples. Depending on the applicationenvisioned, one would desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of the probe orprimers for the target sequence.

[0108] In certain applications, for example, substitution of amino acidsby site-directed mutagenesis, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15M to about 0.9M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results.

[0109] In other embodiments, hybridization may be achieved underconditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mMMgCl2, 10 mM dithiothreitol, at temperatures between approximately 20°C. to about 37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

[0110] E. Primers and Probes

[0111] The term primer, as defined herein, is meant to encompass anynucleic acid that is capable of priming the synthesis of a nascentnucleic acid in a template-dependent process. Typically, primers areoligonucleotides from ten to twenty base pairs in length, but longersequences can be employed. Primers may be provided in double-stranded orsingle-stranded form, although the single-stranded form is preferred.Probes are defined differently, although they may act as primers.Probes, while perhaps capable of priming, are designed to binding to thetarget DNA or RNA and need not be used in an amplification process.

[0112] In other embodiments, the probes or primers are labeled withradioactive species (³²p, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

[0113] One method of using probes and primers of the present inventionis in the search for genes related to Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 or, more particularly,orthologs of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, and SEM A3 from other species. Normally, the target DNA will be agenomic or cDNA library, although screening may involve analysis of RNAmolecules. By varying the stringency of hybridization, and the region ofthe probe, different degrees of homology may be discovered.

[0114] In certain embodiments, it will be advantageous to employ nucleicacids of defined sequences of the present invention in combination withan appropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In otherembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, calorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific, hybridizationwith complementary nucleic acid containing samples.

[0115] Another way of exploiting probes and primers of the presentinvention is in site-directed, or site-specific mutagenesis.Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0116] In general, it is envisioned that the probes or primers describedherein will be useful as reagents in solution hybridization, as in PCR™,for detection of expression of corresponding genes, as well as inembodiments employing a solid phase. Representative solid phasehybridization methods are disclosed in U.S. Pat. Nos. 5,843,663,5,900,481 and 5,919,626. Other methods of hybridization that may be usedin the practice of the present invention are disclosed in U.S. Pat. Nos.5,849,481, 5,849,486 and 5,851,772. The relevant portions of these andother references identified in this section of the Specification areincorporated herein by reference.

[0117] F. Template Dependent Amplification Methods

[0118] A number of template dependent processes are available to amplifythe marker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety. Other methods ofamplication are ligase chain reaction (LCR), Qbeta Replicase, isothermalamplification, strand displacement amplification (SDA), PCR™-liketemplate- and enzyme-dependent synthesis using primers with a capture ordetector moiety, transcription-based amplification systems (TAS),cylical synthesis of single-stranded and double-stranded DNA, “RACE”,one-sided PCR™, and dioligonucleotide amplification.

[0119] Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

[0120] A reverse transcriptase PCR™ amplification procedure may beperformed in order to quantify the amount of mRNA amplified. Methods ofreverse transcribing RNA into cDNA are well known and described inSambrook et al., 1989. Alternative methods for reverse transcriptionutilize thermostable, RNA-dependent DNA polymerases. These methods aredescribed in WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

[0121] G. Vectors

[0122] The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques, which are described in Maniatis et al., 1988 andAusubel et al., 1994, both incorporated herein by reference.

[0123] The term “expression cassette” refers to a vector containing anucleic acid sequence coding for at least part of a gene product capableof being transcribed. In some cases, RNA molecules are then translatedinto a protein, polypeptide, or peptide. In other cases, these sequencesare not translated, for example, in the production of antisensemolecules or ribozymes. Expression vectors can contain a variety of“control sequences,” which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

[0124] H. Promoters and Enhancers

[0125] A “promoter” is a control sequence that is a region of a nucleicacid sequence at which initiation and rate of transcription arecontrolled. It may contain genetic elements at which regulatory proteinsand molecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

[0126] A promoter may be one naturally associated with a gene orsequence, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment and/or exon. Such a promoter canbe referred to as “endogenous.” Similarly, an enhancer may be onenaturally associated with a nucleic acid sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding nucleic acid segmentunder the control of a recombinant or heterologous promoter, whichrefers to a promoter that is not normally associated with a nucleic acidsequence in its natural environment. A recombinant or heterologousenhancer refers also to an enhancer not normally associated with anucleic acid sequence in its natural environment. Such promoters orenhancers may include promoters or enhancers of other genes, andpromoters or enhancers isolated from any other prokaryotic, viral, oreukaryotic cell, and promoters or enhancers not “naturally occurring,”i.e., containing different elements of different transcriptionalregulatory regions, and/or mutations that alter expression. In additionto producing nucleic acid sequences of promoters and enhancerssynthetically, sequences may be produced using recombinant cloningand/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein byreference). Such promoters may be used to drive β-galactosidaseexpression for use as a reporter gene. Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

[0127] Naturally, it will be important to employ a promoter and/orenhancer that effectively directs the expression of the DNA segment inthe cell type, organelle, and organism chosen for expression. Those ofskill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al., (1989), incorporated herein byreference. The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

[0128] Table 2 lists several elements/promoters that may be employed, inthe context of the present invention, to regulate the expression of agene. This list is not intended to be exhaustive of all the possibleelements involved in the promotion of expression but, merely, to beexemplary thereof. Table 3 provides examples of inducible elements,which are regions of a nucleic acid sequence that can be activated inresponse to a specific stimulus. TABLE 2 Promoter and/or EnhancerPromoter/Enhancer References Immunoglobulin Heavy Chain Banerji et al.,1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al.,1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian etal., 1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen et al.,1983; Picard et al., 1984 T-Cell Receptor Luria et al., 1987; Winoto etal., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ β Sullivan et al.,1987 β-Interferon Goodbourn et al., 1986; Fujita et al., 1987; Goodbournet al., 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 ReceptorGreene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989MHC Class II HLA-DRa Sherman et al., 1989 β-Actin Kawamoto et al., 1988;Ng et al.; 1989 Muscle Creatine Kinase Jaynes et al., 1988; Horlick etal., 1989; (MCK) Johnson et al., 1989 Prealbumin (Transthyretin) Costaet al., 1988 Elastase I Omitz et al., 1987 Metallothionein (MTII) Karinet al., 1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987;Angel et al., 1987 Albumin Pinkert et al., 1987; Tronche et al., 1989,1990 α-Fetoprotein Godbout et al., 1988; Campere et al., 1989 t-GlobinBodine et al., 1987; Perez-Stable et al., 1990 β-Globin Trudel et al.,1987 c-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al.,1985 Insulin Edlund et al., 1985 Neural Cell Adhesion Hirsh et al., 1990Molecule (NCAM) α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) HistoneHwang et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A Edbrooke etal., 1989 (SAA) Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Pech et al., 1989 Factor (PDGF) Duchenne Muscular Kiamut et al.,1990 Dystrophy SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh etal., 1985; Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986;Kadesch et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl etal., 1987; Schaffner et al., 1988 Polyoma Swartzendruber et al., 1975;Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981;Dandolo et al., 1983; de Villiers et al., 1984; Hen et al., 1986; Satakeet al., 1988; Campbell and/or Villarreal, 1988 Retroviruses Kriegler etal., 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b,1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al., 1987;Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reismanet al., 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;Spandidos and/or Wilkie, 1983; Spalholz et al., 1985; Lusky et al.,1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987;Stephens et al., 1987; Glue et al., 1988 Hepatitis B Virus Bulla et al.,1986; Jameel et al., 1986; Shaul et al., 1987; Spandau et al., 1988;Vannice et al., 1988 Human Immunodeficiency Muesing et al., 1987; Hauberet al., 1988; Virus Jakobovits et al., 1988; Feng et al., 1988; Takebeet al., 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al.,1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus (CMV)Weber et al., 1984; Boshart et al., 1985; Foecking et al., 1986 GibbonApe Leukemia Holbrook et al., 1987; Quinn et al., 1989 Virus

[0129] TABLE 3 Inducible Elements Element Inducer References MT IIPhorbol Ester (TFA) Palmiter et al., 1982; Heavy metals Haslinger etal., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al.,1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse Glucocorticoids Huang et al., 1981; Lee et al., mammary tumor1981; Majors et al., 1983; virus) Chandler et al., 1983; Lee et al.,1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)xTavernier et al., 1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al.,1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a StromelysinPhorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angelet al., 1987b Murine MX Gene Interferon, Hug et al., 1988 NewcastleDisease Virus GRP78 Gene A23187 Resendez et al., 1988 α-2-MacroglobulinIL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class IGene Interferon Blanar et al., 1989 H-2κb HSP70 E1A, SV40 Large T Tayloret al., 1989, 1990a, Antigen 1990b Proliferin Phorbol Ester-TPA Mordacqet al., 1989 Tumor Necrosis PMA Hensel et al., 1989 Factor ThyroidStimulating Thyroid Hormone Chatterjee et al., 1989 Hormone α Gene

[0130] The identity of tissue-specific promoters or elements, as well asassays to characterize their activity, is well known to those of skillin the art. Examples of such regions include the human LIMK2 gene(Nomoto et al,. 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0131] I. Initiation Signals

[0132] A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

[0133] J. Splicing Sites

[0134] Most transcribed eukaryotic RNA molecules will undergo RNAsplicing to remove introns from the primary transcripts. Vectorscontaining genomic eukaryotic sequences may require donor and/oracceptor splicing sites to ensure proper processing of the transcriptfor protein expression. (See Chandler et al., 1997, herein incorporatedby reference.)

[0135] K. Polyadenylation Signals

[0136] In expression, one will typically include a polyadenylationsignal to effect proper polyadenylation of the transcript. The nature ofthe polyadenylation signal is not believed to be crucial to thesuccessful practice of the invention, and/or any such sequence may beemployed. Specific embodiments include the SV40 polyadenylation signaland/or the bovine growth hormone polyadenylation signal, convenientand/or known to function well in various target cells. Also contemplatedas an element of the expression cassette is a transcriptionaltermination site. These elements can serve to enhance message levelsand/or to minimize read through from the cassette into other sequences.

[0137] L. Origins of Replication

[0138] In order to propagate a vector in a host cell, it may contain oneor more origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

[0139] M. Selectable and Screenable Markers

[0140] In certain embodiments of the invention, the cells containnucleic acid construct of the present invention, a cell may beidentified in vitro or in vivo by including a marker in the expressionvector. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionvector. Generally, a selectable marker is one that confers a propertythat allows for selection. A positive selectable marker is one in whichthe presence of the marker allows for its selection, while a negativeselectable marker is one in which its presence prevents its selection.An example of a positive selectable marker is a drug resistance marker.Examples of selectable and screenable markers are well known to one ofskill in the art.

[0141] N. Host Cells

[0142] In the context of expressing a heterologous nucleic acidsequence, “host cell” refers to a prokaryotic or eukaryotic cell, and itincludes any transformable organisms that is capable of replicating avector and/or expressing a heterologous gene encoded by a vector. A hostcell can, and has been, used as a recipient for vectors. A host cell maybe “transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

[0143] Host cells may be derived from prokaryotes or eukaryotes,depending upon whether the desired result is replication of the vectoror expression of part or all of the vector-encoded nucleic acidsequences. Numerous cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org). An appropriatehost can be determined by one of skill in the art based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5α, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterialcells such as E. coli LE392 could be used as host cells for phageviruses.

[0144] Examples of eukaryotic host cells for replication and/orexpression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,Saos, and PC12. Many host cells from various cell types and organismsare available and would be known to one of skill in the art. Similarly,a viral vector may be used in conjunction with either a eukaryotic orprokaryotic host cell, particularly one that is permissive forreplication or expression of the vector.

[0145] Some vectors may employ control sequences that allow it to bereplicated and/or expressed in both prokaryotic and eukaryotic cells.One of skill in the art would further understand the conditions underwhich to incubate all of the above described host cells to maintain themand to permit replication of a vector. Also understood and known aretechniques and conditions that would allow large-scale production ofvectors, as well as production of the nucleic acids encoded by vectorsand their cognate polypeptides, proteins, or peptides.

[0146] O. Expression Systems

[0147] Numerous expression systems exist that comprise at least a partor all of the compositions discussed above. Prokaryote- and/oreukaryote-based systems can be employed for use with the presentinvention to produce nucleic acid sequences, or their cognatepolypeptides, proteins and peptides. Many such systems are commerciallyand widely available.

[0148] The insect cell/baculovirus system can produce a high level ofprotein expression of a heterologous nucleic acid segment, such asdescribed in U.S. Pat. No. 5,871,986, 4,879,236, both hereinincorporated by reference, and which can be bought, for example, underthe name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUSEXPRESSION SYSTEM FROM CLONTECH®.

[0149] Other examples of expression systems include STRATAGENE®'SCOMPLETE CONTROL™ Inducible Mammalian Expression System, which involvesa synthetic ecdysone-inducible receptor, or its pET Expression System,an E. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

[0150] P. Delivery of Expression Vectors

[0151] There are a number of ways in which expression vectors mayintroduced into cells. In certain embodiments of the invention, theexpression construct comprises a virus or engineered construct derivedfrom a viral genome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

[0152] One of the methods for in vivo delivery involves the use of anadenovirus expression vector. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

[0153] 1.Adenovirus expression vectors

[0154] The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage.

[0155] In one system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0156] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977).

[0157] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

[0158] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0159] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animalstudies suggested that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet and Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz and Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

[0160] 2. Retrovirus expression vectors

[0161] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are, also required for integration in the host cell genome(Coffin, 1990).

[0162] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

[0163] 3. Other viral vectors

[0164] Other viral vectors may be employed as expression constructs inthe present invention. Vectors derived from viruses such as vaccinlavirus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

[0165] In order to effect expression of sense or antisense geneconstructs, the expression construct must be delivered into a cell. Thisdelivery may be accomplished in vitro, as in laboratory procedures fortransforming cells lines, or in vivo or ex vivo, as in the treatment ofcertain disease states. One mechanism for delivery is via viralinfection where the expression construct is encapsidated in aninfectious viral particle.

[0166] 4. Non-viral methods for transfer of expression constructs

[0167] Several non-viral methods for the transfer of expressionconstructs into cultured mammalian cells also are contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

[0168] Once the expression construct has been delivered into the cellthe nucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

[0169] In yet another embodiment of the invention, the expressionconstruct may simply consist of naked recombinant DNA or plasmids.Transfer of the construct may be performed by any of the methodsmentioned above which physically or chemically permeabilize the cellmembrane. This is particularly applicable for transfer in vitro but itmay be applied to in vivo use as well. Dubensky et al. (1984)successfully injected polyomavirus DNA in the form of calcium phosphateprecipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection Benvenistyand Neshif (1986) also demonstrated that direct intraperitonealinjection of calcium phosphate-precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest also may be transferred in a similar manner in vivoand express the gene product.

[0170] In still another embodiment, the transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

[0171] Selected organs including the liver, skin, and muscle tissue ofrats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin etal., 1991). This may require surgical exposure of the tissue or cells,to eliminate any intervening tissue between the gun and the targetorgan, i.e., ex vivo treatment. Again, DNA encoding a particular genemay be delivered via this method and still be incorporated by thepresent invention.

[0172] In a further embodiment of the invention, the expressionconstruct may be entrapped in a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

[0173] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Wong et al., (1980) demonstratedthe feasibility of liposome-mediated delivery and expression of foreignDNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

[0174] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

[0175] Other expression constructs which can be employed to deliver anucleic acid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

[0176] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

[0177] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

[0178] In certain embodiments, gene transfer may more easily beperformed under ex vivo conditions. Ex vivo gene therapy refers to theisolation of cells from an animal, the delivery of a nucleic acid intothe cells in vitro, and then the return of the modified cells back intoan animal. This may involve the surgical removal of tissue/organs froman animal or the primary culture of cells and tissues.

[0179] Primary mammalian cell cultures may be prepared in various ways.In order for the cells to be kept viable while in vitro and in contactwith the expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

[0180] One embodiment of the foregoing involves the use of gene transferto immortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

[0181] Examples of useful mammalian host cell lines are Vero and HeLacells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293,HepG2, NIH3T3, RIN and MDCK cells. In addition, a host cell strain maybe chosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

[0182] A number of selection systems may be used including, but notlimited to, HSV thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosphoribosyltransferase genes,in tk-, hgprt- or apit- cells, respectively. Also, anti-metaboliteresistance can be used as the basis of selection for dhfr, that confersresistance to; gpt, that confers resistance to mycophenolic acid; neo,that confers resistance to the aminoglycoside G418; and hygro, thatconfers resistance to hygromycin.

[0183] Animal cells can be propagated in vitro in two modes: asnon-anchorage dependent cells growing in suspension throughout the bulkof the culture or as anchorage-dependent cells requiring attachment to asolid substrate for their propagation (i.e., a monolayer type of cellgrowth).

[0184] Q. Antibodies

[0185] The antibodies of the present invention are useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Nonionic salts are preferred, sinceother agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations. Antibodies are and their uses arediscussed further below.

[0186] In another aspect, the present invention contemplates an antibodythat is immunoreactive with a Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 molecule of the present invention,or any portion thereof. An antibody can be a polyclonal or a monoclonalantibody. In one embodiment, an antibody is a monoclonal antibody. Meansfor preparing and characterizing antibodies are well known in the art(see, e.g., Howell and Lane, 1988).

[0187] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide of the presentinvention and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a non-humananimal including rabbits, mice, rats, hamsters, pigs or horses. Becauseof the relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

[0188] Antibodies, both polyclonal and monoclonal, specific for isoformsof antigen may be prepared using conventional immunization techniques,as will be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

[0189] It is proposed that the monoclonal antibodies of the presentinvention will find useful application in standard immunochemicalprocedures, such as ELISA and Western blot methods and inimmunohistochemical procedures such as tissue staining, as well as inother procedures which may utilize antibodies specific to Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3-relatedantigen epitopes. Additionally, it is proposed that monoclonalantibodies specific to the particular Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 of different species may beutilized in other useful applications

[0190] In general, both polyclonal and monoclonal antibodies againstFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3 may be used in a variety of embodiments. For example, they may beemployed in antibody cloning protocols to obtain cDNAs or genes encodingother Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, orSEM A3. They may also be used in inhibition studies to analyze theeffects of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 related peptides in cells or animals. Anti-Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 antibodiesalso will be useful in immunolocalization studies to analyze thedistribution of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, and SEM A3 during various cellular events, for example, todetermine the cellular or tissue-specific distribution of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3polypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant Fus 1, 101 F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

[0191] Means for preparing and characterizing antibodies are well knownin the art (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

[0192] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0193] As also is well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0194] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster, injection mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate mAbs.

[0195] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 protein, polypeptide orpeptide or cell expressing high levels of Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3. The immunizing compositionis administered in a manner effective to stimulate antibody-producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep, and frog cells is also possible. The use of ratsmay provide certain advantages (Goding, 1986), but mice are preferred,with the BALB/c mouse being most preferred as this is most routinelyused and generally gives a higher percentage of stable fusions.

[0196] Following immunization, somatic cells with the potential forproducing antibodies, specifically B-lymphocytes (B-cells), are selectedfor use in the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0197] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0198] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, 1986; Campbell, 1984). For example,where the immunized animal is a mouse, one may use P3-X63/Ag8,P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC -11, MPC11- X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3 - Ag1.2.3, IR983F and 4B210; and U-266, GM1500 - GRG2, LICR- LON - HMy2 andUC729 - 6 are all useful in connection with cell fusions.

[0199] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 ratio, though the ratio may vary from about20:1 to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al, (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

[0200] Fusion procedures usually produce viable hybrids, at lowfrequencies, around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0201] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B-cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B-cells.

[0202] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0203] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

[0204] R. Diagnosing Cancers Involving Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3

[0205] Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,and SEM A3 and their corresponding genes may be employed as a diagnosticor prognostic indicator of cancer. More specifically, point mutations,deletions, insertions or regulatory perturbations relating to Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3 maycause cancer or promote cancer development, cause or promoter tumorprogression at a primary site, and/or cause or promote metastasis. Otherphenomena associated with malignancy that may be affected by Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, and SEM A3expression include angiogenesis and tissue invasion.

[0206] 1. Genetic Diagnosis

[0207] One embodiment of the instant invention comprises a method fordetecting variation in the expression of Fus1, 101F6, Gene 21, Gene 26,Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3. This may comprisedetermining that level of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, or SEM A3 or determining specific alterations in theexpressed product. Obviously, this sort of assay has importance in thediagnosis of related cancers. Such cancer may involve cancers of thebrain, lung, liver, spleen, kidney, lymph node, small intestine, bloodcells, pancreas, colon, stomach, cervix, breast, endometrium, prostate,testicle, ovary, skin, head and neck, esophagus, oral tissue, bonemarrow and blood tissue.

[0208] The biological sample can be any tissue or fluid. Variousembodiments include cells of the brain, lung, liver, spleen, kidney,lymph node, small intestine, blood cells, pancreas, colon, stomach,cervix, breast, endometrium, prostate, testicle, ovary, skin, head andneck, esophagus, oral tissue, bone marrow and blood tissue. Otherembodiments include fluid samples such as peripheral blood, lymph fluid,ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid,lacrimal fluid, stool, or urine.

[0209] Nucleic acid used is isolated from cells contained in thebiological sample, according to standard methodologies (Sambrook et al,1989). The nucleic acid may be genomic DNA or fractionated or whole cellRNA. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA. Normally, the nucleic acid is amplified.

[0210] Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

[0211] Following detection, one may compare the results seen in a givenpatient with a statistically significant reference group of normalpatients and patients that have Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3-related pathologies. In this way, itis possible to correlate the amount or kind of Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 detected withvarious clinical states.

[0212] Alterations of a gene include deletions, insertions, pointmutations and duplications. Point mutations result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose occurring in non-germline tissues. Germ-line tissue can occur inany tissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

[0213] A variety of different assays are contemplated in this regard,including but not limited to, fluorescent in situ hybridization (FISH),direct DNA sequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

[0214] 2. Southern/Northern Blotting

[0215] Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

[0216] Briefly, a probe is used to target a DNA or RNA species that hasbeen immobilized on a suitable matrix, often a filter of nitrocellulose.The different species should be spatially separated to facilitateanalysis. This often is accomplished by gel electrophoresis of nucleicacid species followed by “blotting” on to the filter.

[0217] Subsequently, the blotted target is incubated with a probe(usually labeled) under conditions that promote denaturation andrehybridization. Because the probe is designed to base pair with thetarget, the probe will binding a portion of the target sequence underrenaturing conditions. Unbound probe is then removed, and detection isaccomplished as described above.

[0218] 3. Separation Methods

[0219] It normally is desirable, at one stage or another, to separatethe amplification product from the template and the excess primer forthe purpose of determining whether specific amplification has occurred.In one embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

[0220] Alternatively, chromatographic techniques may be employed toeffect separation. There are many kinds of chromatography which may beused in the present invention: adsorption, partition, ion-exchange andmolecular sieve, and many specialized techniques for using themincluding column, paper, thin-layer and gas chromatography (Freifelder,1982).

[0221] 4. Detection Methods

[0222] Products may be visualized in order to confirm amplification ofthe marker sequences. One typical visualization method involves stainingof a gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

[0223] 5. Kit Components

[0224] All the essential materials and reagents required for detectingand sequencing Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™, etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

[0225] 6. RT-PCR™ (Relative Quantitative)

[0226] Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

[0227] In PCR™, the number of molecules of the amplified target DNAincrease by a factor approaching two with every cycle of the reactionuntil some reagent becomes limiting. Thereafter, the rate ofamplification becomes increasingly diminished until there is no increasein the amplified target between cycles. If a graph is plotted in whichthe cycle number is on the X axis and the log of the concentration ofthe amplified target DNA is on the Y axis, a curved line ofcharacteristic shape is formed by connecting the plotted points.Beginning with the first cycle, the slope of the line is positive andconstant. This is said to be the linear portion of the curve. After areagent becomes limiting, the slope of the line begins to decrease andeventually becomes zero. At this point the concentration of theamplified target DNA becomes asymptotic to some fixed value. This issaid to be the plateau portion of the curve.

[0228] The concentration of the target DNA in the linear portion of thePCR™ amplification is directly proportional to the startingconcentration of the target before the reaction began. By determiningthe concentration of the amplified products of the target DNA in PCR™reactions that have completed the same number of cycles and are in theirlinear ranges, it is possible to determine the relative concentrationsof the specific target sequence in the original DNA mixture. If the DNAmixtures are cDNAs synthesized from RNAs isolated from different tissuesor cells, the relative abundances of the specific mRNA from which thetarget sequence was derived can be determined for the respective tissuesor cells. This direct proportionality between the concentration of thePCR™ products and the relative mRNA abundances is only true in thelinear range of the PCR™ reaction.

[0229] The final concentration of the target DNA in the plateau portionof the curve is determined by the availability of reagents in thereaction mix and is independent of the original concentration of targetDNA. Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

[0230] The second condition that must be met for an RT-PCR™ experimentto successfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™experiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for β-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

[0231] Most protocols for competitive PCR™ utilize internal PCR™standards that are approximately as abundant as the target. Thesestrategies are effective if the products of the PCR™ amplifications aresampled during their linear phases. If the products are sampled when thereactions are approaching the plateau phase, then the less abundantproduct becomes relatively over represented. Comparisons of relativeabundancies made for many different RNA samples, such as is the casewhen examining RNA samples for differential expression, become distortedin such a way as to make differences in relative abundances of RNAsappear less than they actually are. This is not a significant problem ifthe internal standard is much more abundant than the target. If theinternal standard is more abundant than the target, then direct linearcomparisons can be made between RNA samples.

[0232] The above discussion describes theoretical considerations for anRT-PCR™ assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR™ is performed as a relative quantitative RT-PCR™with an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

[0233] Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

[0234] One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

[0235] Still other studies may be performed using “real-time” RT-PCR™(Higuchi et al., 1993). These assays detect PCR™ products as theyaccumulate instead of detecting the amount of PCR™ products accumulatedafter a fixed number of cycles. A method of detecting fluorescence aftereach PCR™ cycle is required. The fluorescence signal is plotted versusthe cycle number. The cycle number is expressed as the threshold cycle(C_(T)). The initial fluorescence defines the baseline for the plot andan accumulated PCR™ product is indicated by an increase in fluorescenceabove the baseline. Quantification of the amount of target in a sampleis determined by measuring and comparing the C_(T) to a standard curveto determine the starting copy number.

[0236] “Real-Time” RT-PCR™ (Higuchi et al., 1993) provides more precisequantitation of the amount of target because it is determined during theexponential phase of PCR™, rather than at the endpoint. It also allowshigher throughput because the use of C_(T) values allow a larger dynamicrange. Dilutions of each sample are no longer required.

[0237] 7. Immunodiagnosis

[0238] Antibodies of the present invention can be used in characterizingthe Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,orSEM A3 content of healthy and diseased tissues, through techniques suchas ELISAs and Western blotting. This may provide a screen for thepresence or absence of malignancy or as a predictor of future cancer.

[0239] The use of antibodies of the present invention, in an ELISA assayis contemplated. For example, anti-Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 antibodies are immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

[0240] After binding of antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with the sampleto be tested in a manner conducive to immune complex (antigen/antibody)formation.

[0241] Following formation of specific immunocomplexes between the testsample and the bound antibody, and subsequent washing, the occurrenceand even amount of immunocomplex formation may be determined bysubjecting same to a second antibody having specificity for Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 thatdiffers the first antibody. Appropriate conditions preferably includediluting the sample with diluents such as BSA, bovine gamma globulin(BGG) and phosphate buffered saline (PBS)/Tween®. These added agentsalso tend to assist in the reduction of nonspecific background. Thelayered antisera is then allowed to incubate for from about 2 to about 4hr, at temperatures preferably on the order of about 25° to about 27° C.Following incubation, the antisera-contacted surface is washed so as toremove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween®, or borate buffer.

[0242] To provide a detecting means, the second antibody will preferablyhave an associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease, alkaline phosphatase, glucose oxidase, or(horseradish) peroxidase-conjugated anti-human IgG for a period of timeand under conditions which favor the development of immunocomplexformation (e.g., incubation for 2 hr at room temperature in aPBS-containing solution such as PBS/Tween®).

[0243] After incubation with the second enzyme-tagged antibody, andsubsequent to washing to remove unbound material, the amount of label isquantified by incubation with a chromogenic substrate such as urea andbromocresol purple or 2,2′-azino-di-(3-ethylbenzthiazoline)-6-sulfonicacid (ABTS) and H2O2, in the case of peroxidase as the enzyme label.Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectrum spectrophotometer.

[0244] The preceding format may be altered by first binding the sampleto the assay plate. Then, primary antibody is incubated with the assayplate, followed by detecting of bound primary antibody using a labeledsecond antibody with specificity for the primary antibody.

[0245] The antibody compositions of the present invention will findgreat use in immunoblot or Western blot analysis. The antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. Immunologically-based detection methods for use inconjunction with Western blotting include enzymatically-, radiolabel-,or fluorescently-tagged secondary antibodies against the toxin moietyare considered to be of particular use in this regard.

[0246] 8. Combination of Tumor Suppressors with Other Markers

[0247] Tumors are notoriously heterogeneous, particularly in advancedstages of tumor progression (Morton et al., 1993; Fidler and Hart, 1982;Nowell, 1982; Elder et al., 1989; Bystryn et al., 1985). Although tumorcells within a primary tumor or metastasis all may express the samemarker gene, the level of specific mRNA expression can vary considerably(Elder et al., 1989). It is, in certain instances, necessary to employ adetection system that can cope with an array of heterogeneous markers.

[0248] Thus, while the present invention exemplifies various tumorsuppressors as a markers, any marker that is correlated with thepresence or absence of cancer may be used in combination with thesemarkers to improve the efficacy of tumor detection and treatment. Amarker, as used herein, is any proteinaceous molecule (or correspondinggene) whose production or lack of production is characteristic of acancer cell. Depending on the particular set of markers employed in agiven analysis, the statistical analysis will vary. For example, where aparticular combination of markers is highly specific for melanomas orbreast cancer, the statistical significance of a positive result will behigh. It may be, however, that such specificity is achieved at the costof sensitivity, i.e., a negative result may occur even in the presenceof melanoma or breast cancer. By the same token, a different combinationmay be very sensitive, i.e., few false negatives, but has a lowerspecificity.

[0249] As new markers are identified, different combinations may bedeveloped that show optimal function with different ethnic groups orsex, different geographic distributions, different stages of disease,different degrees of specificity or different degrees of sensitivity.Marker combinations also may be developed, which are particularlysensitive to the effect of therapeutic regimens on disease progression.Patients may be monitored after surgery, gene therapy, hyperthermia,immunotherapy, cytokine therapy, gene therapy, radiotherapy orchemotherapy, to determine if a specific therapy is effective.

[0250] One particularly useful combination of markers for melanoma istyrosinase and members of the MAGE family, particularly MAGE-1 orMAGE-3. Human tyrosinase is an essential enzyme which regulates theproduction of melanin (Nordlund et al., 1989; Hoon et al., 1993), agroup of brown or black pigments in the skin and eyes of humans. Morespecifically, tyrosinase catalyzes the conversion of tyrosine to Dopaand of Dopa to dopaquinone.

[0251] There are many other markers that may be used in combination withthese, and other, markers. For example, b-human chorionic gonadotropin(b-HCG). b-HCG is produced by trophoblastic cells of placenta ofpregnant woman and is essential for maintenance of pregnancy at theearly stages (Pierce et al., 1981; Talmadge et al., 1984). b-HCG isknown to be produced by trophoblastic or germ cell origin tumors, suchas choriocarcinoma or testicular carcinoma cells (Madersbacher et al.,1994; Cole et al., 1983). Also ectopic expression of b-HCG has beendetected by a number of different immunoassays in various tumors ofnon-gonadal such as breast, lung, gastric, colon, and pancreas, etc.(McManus et al., 1976; Yoshimura et al., 1994; Yamaguchi et al., 1989;Marcillac et al., 1992; Alfthan et al., 1992). Although the function ofb-HCG production in these tumors is still unknown, the atavisticexpression of b-HCG by cancer cells and not by normal cells ofnon-gonadal origin suggests it may be a potentially good marker in thedetection of melanoma and breast cancer (Hoon et al., 1996; Sarantou etal., 1997).

[0252] Another exemplary example of a marker is glycosyltransferase b-1,4-N-acetylgalacto-saminyltransferase (GalNAc). GalNAc catalyzes thetransfer of N-acetylgalactosamine by b1(r) 4 linkage onto bothgangliosides GM3 and GD3 to generate GM2 and GD2, respectively (Nagataet al., 1992; Furukawa et al., 1993). It also catalyzes the transfer ofN-acetylgalactosamine to other carbohydrate molecules such as mucins.Gangliosides are glycosphingolipids containing sialic acids which playan important role in cell differentiation, adhesion and malignanttransformation. In melanoma, gangliosides GM2 and GD2 expression, areoften enhanced to very high levels and associate with tumor progressionincluding metastatic tumors (Hoon et al., 1989; Ando et al., 1987;Carubia et al., 1984; Tsuchida et al., 1987a). Gangliosides are alsoexpressed in melanoma, renal, lung, breast carcinoma cancer cells. Thegangliosides GM2 and GD2 are immunogenic in humans and can be used as atarget for specific immunotherapy such as human monoclonal antibodies orcancer vaccines (Tsuchida et al., 1987b; Irie, 1985.)

[0253] GalNAc mRNA may be used as a marker of GM2 and GD2 expression andconsequently a marker of either melanoma or breast cancer cells. GalNAcis generally not expressed in normal lymphocytes, epithelial cells,melanocytes, connective tissue or lymph node cells. If detected, it isin very low levels. Prostate specific antigen is a well characterizedmarker for prostate cancer (Gomella et al., 1997). bcr/abl gene forleukemia is a further well characterized marker that is contemplated tobe useful in combination with HOJ-1.

[0254] Other markers contemplated by the present invention includecytolytic T lymphocyte (CTL) targets. MAGE-3 is a marker identified inmelanoma cells and breast carcinoma. MAGE-3 is expressed in manymelanomas as well as other tumors and is a (CTL) target (Gaugler et al.,1994). MAGE-1, MAGE-2, MAGE-4, MAGE-6, MAGE-12, MAGE-Xp, and are othermembers of the MAGE gene family. MAGE-1 gene sequence shows 73% identitywith MAGE-3 and expresses an antigen also recognized by CTL (Gaugler etal., 1994). MART-1 is another potential CTL target (Robbins et al.,1994) and also may be included in the present invention.

[0255] MUC18 is another marker that is useful in the identification ofmelanoma cells (Lehman et al, 1989; Lehman et al., 1987). MUC18 is acell surface glycoprotein that is a member of the immunoglobulinsuperfamily and possesses sequence homology to neural cell adhesionmolecules (NCAM). Other mucin family members include MUC1, MUC2, MUC3and MUC4. These were found to be expressed at a high level in certaintumor cell lines (Hollingsworth et al., 1994) and also may be used asmarkers in combination with the novel HOJ-1 marker of the presentinvention.

[0256] Other members of the immunoglobulin superfamily of adhesionmolecules associated with the development of melanoma metastasis (Dentonet al., 1992) may be utilized in the present invention. Examples includeintercellular adhesion molecule-1 (ICAM-1) NCAM, VCAM-1 and ELAM.Another embodiment includes carcinoma cell related molecules andmolecules associated with other metastatic diseases such ascarcinoembryonic antigen (CEA; Lin and Guidotti, 1989).

[0257] Other carcinoma or skin cancer associated proteins and theircorresponding nucleic acids also may be utilized in the presentinvention. Preferred examples include melanoma antigen gp75(Vijayasardahi et al., 1990), human cytokeratin 20, high molecularweight melanoma antigen (Natali et al., 1987) and cytokeratin 19 (K19)(Datta et al., 1994). Other markers that may be useful herein includeinhibitors of the cyclin-dependent kinases, (CDK). For example, CDK4regulates progression through the G1 phase of the cell cycle. Theactivity of CDK4 is controlled by an activating subunit, D-type cyclin,and by an inhibitory subunit, the p16INK4 has been biochemicallycharacterized as a protein that specifically binds to and inhibits CDK4(Serrano et al., 1993; Serrano et al., 1995). Other CDK-inhibitoryproteins that also includes p16, p21WAF1, and p27KIP1. This list is notintended to be exhaustive, but merely exemplary, for the type and numberof potential markers which may be used in the present invention.

[0258] Other proteins and their corresponding nucleic acids related tothe melanin synthesis pathway may be used as markers, such as tyrosinaserelated protein 1 and 2 and members of the pMel 17 gene family (Kwon etal., 1993).

[0259] Preferred embodiments of the invention involve many differentcombinations of markers for the detection of cancer cells. Any markerthat is indicative of neoplasia in cells may be included in thisinvention.

[0260] S. Transgenic Animals/Knockout Animals

[0261] In one embodiment of the invention, transgenic animals areproduced which contain a functional transgene encoding a functionalFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3 polypeptide or variants thereof. Transgenic animals expressing Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3. Transgenicanimals of the present invention also can be used as models for studyingindications such as cancers.

[0262] In one embodiment of the invention, a Fus1, 101F,6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 transgene is introducedinto a non-human host to produce a transgenic animal expressing a humanor murine Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6,123F2, or SEM A3 gene. The transgenic animal is produced by theintegration of the transgene into the genome in a manner that permitsthe expression of the transgene. Methods for producing transgenicanimals are generally described by Wagner and Hoppe (U.S. Pat. No.4,873,191; which is incorporated herein by reference), Brinster et al.1985; which is incorporated herein by reference in its entirety) and in“Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds.,Hogan, Beddington, Costantimi and Long, Cold Spring Harbor LaboratoryPress, 1994; which is incorporated herein by reference in its entirety).

[0263] It may be desirable to replace the endogenous Fus1, 101F6, Gene21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, a Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 gene flanked by genomic sequences istransferred by microinjection into a fertilized egg. The microinjectedeggs are implanted into a host female, and the progeny are screened forthe expression of the transgene. Transgenic animals may be produced fromthe fertilized eggs from a number of animals including, but not limitedto reptiles, amphibians, birds, mammals, and fish. Within a particularembodiment, transgenic mice are generated which overexpress Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 or expressa mutant form of the polypeptide. Alternatively, the absence of a Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 in“knock-out” mice permits the study of the effects that loss of Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3protein has on a cell in vivo. Knock-out mice also provide a model forthe development of Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3-related cancers.

[0264] As noted above, transgenic animals and cell lines derived fromsuch animals may find use in certain testing experiments. In thisregard, transgenic animals and cell lines capable of expressingwild-type or mutant Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3 may be exposed to test substances. These testsubstances can be screened for the ability to enhance wild-type Fus1,10F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3expression and or function or impair the expression or function ofmutant Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3.

[0265] Promoter sequences mentioned within this document may be used todrive β-galactosidase expression. The use of a β-galactosidase reporterconstruct in transgenic mice may be used to identify factors whichregulate Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 expression.

[0266] T. Methods for Treating Cancers Using Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3

[0267] The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement of Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3. Byinvolvement, it is not even a requirement that Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 be mutated orabnormal—the overexpression of Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 may actually overcome other lesionswithin the cell. Thus, it is contemplated that a wide variety of cancercells may be treated using Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1,Luca2, PL6, 123F2, or SEM A3 therapy, including brain, lung, liver,spleen, kidney, lymph node, small intestine, blood cells, pancreas,colon, stomach, cervix, breast, endometrium, prostate, testicle, ovary,skin, head and neck, esophagus, oral tissue, bone marrow and bloodtissue.

[0268] In many contexts, it is not necessary that the cancer cell bekilled or induced to undergo normal cell death or “apoptosis.” Rather,to accomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis partially or completely blocked, however, or that some tumorregression is achieved. Clinical terminology such as “remission” and“reduction of tumor” burden also are contemplated given their normalusage.

[0269] 1. Genetic Based Therapies

[0270] One of the therapeutic embodiments contemplated by the presentinventors is the intervention, at the molecular level, in the eventsinvolved in the tumorigenesis of some cancers. Specifically, the presentinventors intend to provide, to a cancer cell, an expression cassettecapable of providing Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3 to that cell. The lengthy discussion of expressionvectors and the genetic elements employed therein is incorporated intothis section by reference. Particularly preferred expression vectors areviral vectors such as adenovirus, adeno-associated virus, herpesvirus,vaccinia virus and retrovirus. Also preferred isliposomally-encapsulated expression vector.

[0271] Various routes are contemplated for various tumor types. Thesection below on routes contains an extensive list of possible routes.For practically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector. A tumor bedmay be treated prior to, during or after resection. Following resection,one generally will deliver the vector by a catheter left in placefollowing surgery. One may utilize the tumor vasculature to introducethe vector into the tumor by injecting a supporting vein or artery. Amore distal blood supply route also may be utilized.

[0272] In a different embodiment, ex vivo gene therapy is contemplated.This approach is particularly suited, although not limited, to treatmentof bone marrow associated cancers. In an ex vivo embodiment, cells fromthe patient are removed and maintained outside the body for at leastsome period of time. During this period, a therapy is delivered, afterwhich the cells are reintroduced into the patient; hopefully, any tumorcells in the sample have been killed.

[0273] 2. Protein Therapy

[0274] Another therapy approach is the provision, to a subject, of Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3polypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

[0275] 3. Combined Therapy with Immunotherapy, Traditional Chemo- orRadiotherapy

[0276] Tumor cell resistance to DNA damaging agents represents a majorproblem in clinical oncology. One goal of current cancer research is tofind ways to improve the efficacy of chemo- and radiotherapy. One way isby combining such traditional therapies with gene therapy. For example,the herpes simplex-thymidine kinase (HS-tk) gene, when delivered tobrain tumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated that Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3replacement therapy could be used similarly in conjunction with chemo-or radiotherapeutic intervention. It also may prove effective to combineFus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEMA3 gene therapy with immunotherapy, as described above.

[0277] To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a Fus1,101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PI,6, 123F2, or SEM A3expression construct and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the expression construct and the agent(s) or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

[0278] Alternatively, the gene therapy treatment may precede or followthe other agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

[0279] It also is conceivable that more than one administration ofeither Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 or the other agent will be desired. Various combinations maybe employed, where Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2,PL6, 123F2, or SEM A3 is “A” and the other agent is “B”, as exemplifiedbelow: A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/BA/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/AA/A/B/A A/B/B/B B/A/B/B B/B/A/B

[0280] Other combinations are contemplated. Again, to achieve cellkilling, both agents are delivered to a cell in a combined amounteffective to kill the cell.

[0281] Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, acceleratedprotons, UV-irradiation, microwaves, electronic emissions, and the like.A variety of chemical compounds, also described as “chemotherapeuticagents,” function to induce DNA damage, all of which are intended to beof use in the combined treatment methods disclosed herein.Chemotherapeutic agents contemplated to be of use, include, e.g.,cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, famesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate and evenhydrogen peroxide. The invention also encompasses the use of acombination of one or more DNA damaging agents, whether radiation-basedor actual compounds, such as the use of X-rays with cisplatin or the useof cisplatin with etoposide. In certain embodiments, the use ofcisplatin in combination with a Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3 expression construct is particularlypreferred as this compound.

[0282] In treating cancer according to the invention, one would contactthe tumor cells with an agent in addition to the expression construct.This may be achieved by irradiating the localized tumor site withradiation such as X-rays, accelerated protons, UV-light, γ-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with theagent by administering to the subject a therapeutically effective amountof a pharmaceutical composition comprising a compound such as,adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D,mitomycin C, or more preferably, cisplatin. The agent may be preparedand used as a combined therapeutic composition, or kit, by combining itwith a Fus1, 101F6, Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2,or SEM A3 expression construct, as described above.

[0283] Agents that directly cross-link nucleic acids, specifically DNA,are envisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with Fus1, 101 F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3. Agents such as cisplatin, and otherDNA alkylating agents may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

[0284] Agents that damage DNA also include compounds that interfere withDNA replication, mitosis and chromosomal segregation. Suchchemotherapeutic compounds include adriamycin, also known asdoxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widelyused in a clinical setting for the treatment of neoplasms, thesecompounds are administered through bolus injections intravenously atdoses ranging from 25-75 mg/m² at 21 day intervals for adriamycin, to35-50 mg/m² for etoposide intravenously or double the intravenous doseorally.

[0285] Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

[0286] Other factors that cause DNA damage and have been usedextensively include what are commonly known as γ-rays, X-rays,accelerated protons, and/or the directed delivery of radioisotopes totumor cells. Other forms of DNA damaging factors are also contemplatedsuch as microwaves, and UV-irradiation. It is most likely that all ofthese factors effect a broad range of damage DNA, on the precursors ofDNA, the replication and repair of DNA, and the assembly and maintenanceof chromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 weeks), to singledoses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes varywidely, and depend on the half-life of the isotope, the strength andtype of radiation emitted, and the uptake by the neoplastic cells.

[0287] The skilled artisan is directed to “Remington's PharmaceuticalSciences” 15th Edition, chapter 33, in particular pages 624-652. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics Standards.

[0288] The inventors propose that the regional delivery of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3 expressionconstructs to patients with 3p21.3-linked cancers will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease. Similarly, the chemo- or radiotherapymay be directed to a particular, affected region of the subjects body.Alternatively, systemic delivery of expression construct and/or theagent may be appropriate in certain circumstances, for example, whereextensive metastasis has occurred.

[0289] In addition to combining Fus1, 101F6, Gene 21, Gene 26, Beta*,Luca1, Luca2, PL6, 123F2, or SEM A3-targeted therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of Fus1, 101F6,Gene 21, Gene 26, Beta*, Luca1, Luca2, PI,6, 123F2, or SEM A3 and p53 orp16 mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf,erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl.

[0290] It also should be pointed out that any of the foregoing therapiesmay prove useful by themselves in treating a Fus1, 101F6, Gene 21, Gene26, Beta*, Luca1, Luca2, PL6, 123F2, or SEM A3-related disorder. In thisregard, reference to chemotherapeutics and non-Fus1, 101F6, Gene 21,Gene 26, Beta*, Luca1, Luca2, PL6, l23F2, or SEM A3 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

[0291] 4. Formulations and Routes for Administration to Patients

[0292] Where clinical applications are contemplated, it will benecessary to prepare pharmaceutical compositions—expression vectors,virus stocks, proteins, antibodies and drugs—in a form appropriate forthe intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

[0293] One will generally desire to employ appropriate salts and buffersto render delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

[0294] The active compositions of the present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions. Upon formulation, solutions will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective.

EXAMPLES

[0295] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those ofskilled the art that the techniques disclosed in the examples whichfollow represent techniques discovered by the inventor to function wellin the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

EXAMPLE 1 Identification of 3p Genes in 3p21.3 Critical Region andIsolation of cDNA of 3p Genes

[0296] The 3p tumor suppressor region was identified by allelotypingdesigned to search for areas of LOH in matched tumor/normal tissuepairs, and examine uncommon examples of homozygous deletions (FIG. 4).The most frequently involved region showing allele loss in lung cancerwas mapped to the 3p21.3 region. Furthermore, multiple overlappinghomozygous deletions have been found in the 3p21.3 chromosome region inSCLC lines H740 and H1450, which narrowed down the search for the tumorsuppressor genes flanking about 750 kb in 3p21.3 region. Nine genes,Fus1, 101F6, NPRL2 (Gene 21), CACNA2D2(Gene 26), HYAL1 (Luca 1), HYAL2(Luca 2), PL6, 123F2, and Beta*, were either disrupted or immediatelyflanking a ˜35 kb homozygous deletion found in the 3p21.3 region. SEM A3is also present in the 3p21.3 region. The cDNAs of these genes wereisolated and cloned, and mutations in these genes were determined invarious tumor and tumor cell lines by single strand conformationpolymorphism (SSCP) and DNA sequencing analysis (Table 4). Some of thecDNA clones showed ˜50% amino acid homologies to known genes in theGeneBank, some demonstrated complete DNA sequence homology to randomsequence tagged sites in the GeneBank, and one gene, Beta*, waspreviously unknown (Table 4). TABLE 4 Genes Identified in the 125 kb3p21.3 Critlical Region and Status of Their cDNA Sequencing and MutationAnalysis GenBAnk CDNA Sequence (bp) Mutation Analysis Gene* Number (aa)(Numbers) Mutations** CACNA2D2 (Gene 26) AF040709 5,482 bp (1,205 aa)Yes (60) none PL6 U09584 1,860 bp (351 aa) Yes (38) none 101F6 AF0407091,117 bp (222 aa) Yes (38) none NPRL2 (Gene 21) AF040707, 1,696 bp (203aa) Yes (38) 1 stop AF040708 Beta* (BLU) none 1,739 bp (440 aa) Yes (61)3 missense 123F2 (RASSF1) AF040703 1,502 bp (431 aa) Yes (37) none FUS-1AF055479 1,696 bp (161 aa) Yes (79) 2 stop HYAL2 (LUCA-2) U09577 1,783bp (473 aa) Yes (40) none HYAL1 (LUCA-1) U03056 2,565 bp (435 aa) Yes(40) 2 missense

EXAMPLE 2 Construction of Recombinant Adenoviral Vectors of 3p Genes

[0297] Adenoviral vectors have been widely used for gene delivery invitro, in animal models, preclinical research, and human clinical genetherapy trials. The high efficiency of transduction and high-levelexpression of transgenes mediated by adenovirus vectors in various celltypes are reasons why the recombinant adenoviral vectors expressing the3p genes (Ad-3ps) are effective tools for introduction of the functionalwild-type 3p genes into tumors or tumor cell lines with abnormalities of3p or 3p genes.

[0298] Recombinant adenoviral vectors of 3p21 genes, including Gene2l,Fus1, 101F6, Gene 26, 123F2S, Luca1, and Beta*, have been constructedusing the inventors' recently developed ligation-mediatedplasmid-adenovirus vector construction system, pAd-RAP andpAd-RAP-Shuttle. The inventors have successfully and rapidly constructedrecombinant adenoviral vectors for all ten genes in the 3p21.3 regionand many other recombinant vectors using this system. Recombinant Ad-3pscan efficiently deliver 3p genes into and express them in various celltypes in vitro by directly infecting target cells and in vivo byintravenous or local injection of vectors. The relative genomiclocations of the tumor suppressor 3p21.3 genes in chromosome 3p and thestructure of the recombinant adenoviral vectors of 3p genes areschematically demonstrated (FIG. 5).

[0299] The inventors have developed a novel ligation-mediatedplasmid-adenovirus vector construction system, named pAd-RAP andpAd-RAP-Shuttle. This system can be used to rapidly constructrecombinant adenovirus-containing plasmids in bacterial Escherichiacoli, and then successfully produce homogeneous adenovirus in mammalianhost 293 cells (FIG. 1). In this system, the transgene (X) is firstplaced in a plasmid shuttle vector, pAd-RAP-Shuttle, containing theadenoviral inverted repeated terminal (IRT) sequence, an expressioncassette of a cytomegalovirus (CMV) promoter and bovine growth hormone(BGH) poly (A) signal sequence, and two unique restriction sites BstBIand ClaI at the 5′ and 3′ends of the IRT-CMV-multiple cloning sites-BGHsequence, respectively. The BstBI/ClaI-released DNA fragment containingIRT-CMV-X-BGH is then inserted into an adenoviral plasmid vector,pAd-RAP, which contains a complete E1 and E3-deleted adenovirus type 5genome and three unique restriction sites (PacI, BstBI, and ClaI), by invitro ligation using BstBI and ClaI sites. After transformation intoEscherichia coli, 90% of the transformants have the correct insert.Finally, PacI/BstBI digestion of the resulting plasmid allows release ofthe entire adenovirus genome-containing the 3p gene. The recombinantAd-X DNA is then transfected into 293 cells, resulting in a homogeneouspopulation of recombinant Ad-X. Other promoters, poly A sequences, andrestriction sites (can be used. This system can be used to rapidlyconstruct a recombinant adenovirus within 2-4 weeks. By comparison, theconventional methods, such as that using homologous recombination inmammalian cells, will usually take 3-12 months.

[0300] In case of failure to produce a specific recombinant Adenovirusdue to the possible cytotoxicity of the transgenes in the host 293cells, a similar system named pAd-RAP-TetR-Off andpAd-RAP-TRE-CMV-Shuttle, as demonstrated in FIG. 2, with tetracyclineregulatory elements (TRE) that can turn off transgene expression in thepresence of tetracycline has been developed and can be used for theproduction of such vectors.

[0301] The Ad-CMV-GFP (Ad-GFP) and Ad-CMV-LacZ vectors were used tomonitor transduction efficiency by the viral vectors and as nonspecifictransgene expression controls. Ad-E1- (Ad-EV), an empty E1- vector, isused as a negative control. Viral titers were determined by both opticaldensity measurement and plaque assay. Potential contamination of theviral preparation by the wild-type virus was monitored by polymerasechain reaction (PCR) analysis. Sequences of 3p genes in the viralvectors were confirmed by automated DNA sequencing. The resulting Ad-3psare named, Ad-101F6, Ad-Gene26, Ad-Gene2l, Ad-Fus1, Ad-PL6, Ad-Luca1,Ad-Luca2, Ad-123F2S, Ad-Beta*, and Ad-SEM A3, respectively.

EXAMPLE 3 Preparation of PAD3ps

[0302] The preparation of protamine-adenovirus (PAD) complexes andenhancement of transduction efficiency by PADs in vitro and in vivo havebeen reported^(47,48). The protamine-adenovirus complexes were preparedby simply mixing 1×10¹⁰ viral particles with 50 μg of protamine sulfate(10 mg/ml). The complexes were incubated for 10 min at room temperature,and then the complexed adenovirus were diluted in an appropriate volumeof PBS for designated in vitro or in vivo experiments.

EXAMPLE 4 Preparations of LDC3ps and LPD3ps

[0303] The liposome (DOTAP:Cholesterol) (LDC), plasmid DNA, and LDC-3pDNA complexes (LDC3ps) were prepared as described by Templeten et al.⁴⁹.LDC3ps were formulated as 80 nmol liposome:50 μg DNA in 5% Dextral water(D5W) at a total volume of 100 μl for intravenous injection to onemouse. Liposome (DOTAP:Cholesterol):Protamine:DNA (LPD) were preparedbased on the method of Hung⁵⁰.

EXAMPLE 5 Effects of overexpression of 3p genes on Tumor Cell Growth

[0304] To study biological function of new tumor suppressor genes,experiments are conventionally performed in tumor cell lines eithertransiently or stably transfected by wild type gene-expressing plasmids.The Ad-3p vectors can offer several advantages over plasmids for 3p genedelivery in vitro and in vivo: 1) high efficiency (>80%) of transductionand high level of 3p gene expression can be easily achieved in a widespectrum of cell types by simply adjusting the multiplicity of infection(MOI) of viral particles to target cells, consequently, the Ad-3ps canbe used to evaluate effects of 3p genes as a individual or as a wholeregion 2) Ad-3ps-transduction can be directly applied to tumor cells tostudy their effect on tumorigenicity in animals without selection ofstably transduced colonies, by which problems associated with colonyselection process and unknown effectors or factors generated inresulting cell colonies can be avoided; and 3) Ad-3ps can be directlyused to evaluate the role of 3p genes as a tumor suppressor gene regionin vivo by either intravenous or intratumoral injection of animals withthe individual or combined Ad-3p vectors.

[0305] The biological function of these newly isolated 3p genes ischaracterized in this invention by liposome- and recombinant adenoviralvector-mediated gene transfer both in vitro and in vivo. Human lungcancer cell lines (H1299, H358, H460, and A549), with varied status ofchromosome 3p or individual 3p genes and a normal human bronchialepithelial cell (HBEC) line were used to evaluate the effects of 3pgenes on cell growth arrest, proliferation, apoptosis, and cell cyclekinetics in vitro and on growth of the primary and metastatic tumors inanimal models.

[0306] To test the hypothesis that the 3p genes function as tumorsuppressors in vitro, the inventors performed a series of experiments tostudy the effects of overexpression of the 3p genes on cellproliferation in various human non-small cell lung cancer cells and anormal human bronchial epithelial cell line (HBEC) varying in status of3p chromosomal structure or genes and gene products (Table 5) byliposome- or adenoviral vector-mediated 3p gene transfer. One of theselines is H1299, a NSCLC cell line that contains an internal homozygousdeletion of p53, and has no normal copy of chromosome 3 with LOH of 3palleles and has very high levels of telomerase expression and activity.A549, is a lung carcinoma cell line that contains wild-type p53 withabnormal 3p alleles; H358 is a lung cancer cell line that containswild-type p53 with two 3p alleles; and H460 is, a lung cancer cell linethat contains wild-type p53 with loss of noe allele of the 3p21.3 region(Table 5). Normal HBECs or fibroblast cells (Clonetics Inc.,Walkersville, Md.) were also used to evaluate the general toxicity ofthe 3p genes and Ad-3ps. The 293 cell line was used in the construction,amplification, and titration of adenoviral vectors. The cells weremaintained in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/lof glucose with 10% FBS. TABLE 5 Status of 3p Genes in Human Lung CancerCell Lines and Normal HBEC* Cell Line Origin 3p Genes P53 hTERT H1299Lung, large LOH Deletion High Activity A549 NSCL LOH WT Active H460Lung, Large LOH WT Active H358 NSCL WT Deletion Active HBEC BronchialEpithelia Normal Normal undetected

[0307] The Ad-3p vectors, protamine-Ad-3p complexes (PAD3ps) or liposome(DOTAP)-3p plasmid DNA complexes (LPD3ps) developed in this inventioncan be used to deliver 3p genes efficiently to the tumor cells in vitroby direct transduction and to the primary and distant lung or othermetastatic tumor sites in vivo by systemic administration. Thespontaneous or experimental pulmonary metastasis models of human lungcancers H1299 and A549, as well as other cancers, can be used to studythe effects of 3p genes on tumor progression and metastasis by systemictreatment of lung metastatic tumors in mice through intravenousinjection of either PAD3p or LPD3p complexes.

[0308] In experiments with liposome-mediated 3p gene transfer in H1299cells, six genes out of the nine, Fus1, 101F6, Luca1, 123F2S, Beta*, andGene 21, demonstrated varied degrees (20-65%) of cell growth inhibitionin H1299 transfectants after 48 hr of transfection, compared tountransfected and empty CMV vector-transfected controls. Three othergenes Gene 26, PL6, and Luca 2 showed no significant effects on H1299cell growth under the same experimental condition (Table 6). Theobserved inhibitory effect of Fus1, Beta*, 123F2S, and Gene 21 on H1299cell growth were comparable to that of highly cytotoxic gene Bak underthe same experiment conditions (Table 6). The three other genes Gene 26,PL6, and Luca 2 showed no significant effects on H1299 cell growth underthe same experimental conditions. Varied degrees (10-40%) of inductionof apoptosis and altered cell cycle kinetics (changes of cellpopulations at G0, G1 and S phases) were observed in H1299 cellstransfected with plasmids containing genes Fus1, 101F6, 123F2S, Luca,and Beta* by FACS analysis with TUNEL reaction and PI staining. TABLE 6Effects of DOTAP-mediated 3p Gene Expression on Growth of H1299 Cells(48 h) Data from MDACC Data from UTSMC (Transfection) (% Cell (ColonyFormation)^(†) Viability ± STDEV) (% Cell Viability ± STDEV) PBS 100 NDCMV-EV 85 ± 9.5 100 GFP 84 ± 7.2 ND Bak 54 ± 5.1 ND 101F6 76 ± 3.5 52 ±10.0 Fus1 78 ± 2.1 49 ± 14.0 Gene 21 45 ± 6.5 83.7 ± 17.7   Gene 26  88± 12.5 40 ± 0.00 Luca 1 81 ± 2.8 66 ± 27.2 Luca 2 100 ± 9.8  80 ± 27.6PL6 100 ± 13.6 95 ± 53.8 123F2S 67 ± 3.8 58 ± 0.0  Beta* 35 ± 2.3 51 ±8.5 

[0309] Effects of 3p genes on tumor cell growth were furthercharacterized by recombinant-adenoviral vector-mediated 3p gene transferin various lung cancer cell lines and a normal HBEC line. To test thespecificity of the observed inhibitory effects of 101F6, Fus1, andGene21 overexpression on tumor cell proliferation and the potentialcytotoxicity of the overexpressed 3p genes, the inventors analyzed theeffect of these 3p genes on cell proliferation in Ad-3p-transduced wildtype 3p-containing H358 cells and normal HBEC cells (FIG. 6). Cells ineach line were transduced in vitro by Ad-101F6, Ad-fus1 and Ad-Gene21vectors administered at various multiplicity of infections (MOIs) inviral particles/cell (vp/c); cells treated with PBS were used as mock,empty vector Ad-EV as negative, Ad-LacZ as nonspecific, and Ad-p53 aspositive controls, respectively. A less than 10% loss of cell viabilityin Ad-3p-transduced HBEC and a less than 20% loss in H358 cells atvarious MOIs, were observed when compared with that in untransducedcontrol cells. Similar losses were also observed in Ad-EV- and Ad-LacZtransduced cells and slightly higher loses in Ad-p53-transduced cellsthroughout the posttransduction time course, suggesting that nogeneralized cytotoxicity was associated with overexpression of these 3pgenes. The transduction efficiency was determined by examining theGFP-expressing cells in the Ad-GFP transduced cell population under afluorescence microscope.

[0310] The transduction efficiency of the adenoviral vectors was greaterthan 80% at the highest MOI applied for each cell line. Cellproliferation was analyzed by determining the viability of cells at 1, 3and 5 days posttransduction, respectively. Cell viability wassignificantly reduced in Ad-101F6, Ad-Fus1, and Ad-Gene2l transducedA549 and H460 cells which exhibit LOH in 3p region but contain wild-typep53 and H1299 cells which contains homozygous deletions of 3p region andp53 (FIG. 6). In all cases, the viability of transduced cells wascompared with that of untransduced (PBS-treated) control cells (whoseviability was set at 100%).

[0311] The overexpression of 3p genes in these Ad-3p tranfectants wasverified by a quantitative Real Time RT-PCR, and known concentrations ofhuman total RNA, primers, and TaqMan probes for β-actin DNA were used asstandards and as a internal control (FIG. 7). TaqMan probes and primersof 3p genes were designed using a Primerexpress software (Perkin ElmerApplied Biosystems, Foster City, Calif.). Human genomic DNA or totalRNAs were used as template standards and human β-actin orglyceraldehyde-3-phosphate dehydrogenase (GAPDH) TaqMan probes andprimers as controls. Total RNA was isolated from Ad-3p transduced tumorcells or tumor specimen using TRIZOL. Real time RT-PCR andquantification of RT-PCR products were performed and analyzed using aTaqMan Gold RT-PCR Kit, an ABI Prism 7700 Sequence Detection System andequipped software. These results show that overexpression of these 3pgenes can inhibit tumor cell growth in vitro.

EXAMPLE 6 Effects of 3p Genes on Tumor Cell Growth and Proliferation

[0312] To test whether the growth properties of various lung cancercells with abnormalities of 3p or 3p genes could be altered by theintroduction of wild-type 3p genes, cell viability in Ad-3p-transducedtumor cells at varied MOIs at designated posttransduction time intervalsare assayed by XTT staining as described previously,⁴⁴ and theuntransduced and Ad-EV-, Ad-GFP-, or Ad-LacZ-transduced cells were usedas controls. Each experiment was repeated at least three times, witheach treatment given in duplicate or triplicate. Proliferation of theAd-3p-transduced cells were analyzed by animmunofluorescence-enzyme-linked immunosorbent assay for incorporationof bromodeoxyuridine (BrdU) into cellular. Ad-3p-transduced normal HBECswere used to evaluate the possible general toxicity of the 3p genes andAd-3ps in vitro. Transcription and expression of 3p genes inAd-3p-transduced cells were examined by reverse transcriptase-polymerasechain reaction or Northern- and Western-blot analysis with anti-3pprotein polyclonal antibodies.

EXAMPLE 7 Western Blot Analysis of Expression of 3p (Genes inAd-3p-transduced Cells

[0313] Expression of 3p genes in Ad-3p-transduced cells was analysed byWestern blot, using polyclonal antibodes aganist polypeptides derivedfrom predicted 3p amino acid sequences or monoclonal antibodies againstc-myc of FLAG tags in 3p fusion proteins. Cells grown in 60 mm-dishes(1-5×10⁶/well) were treated with Ad-3ps, and PBS alone was used as acontrol. Proteins were separated by SDS-PAGE. Each lane was loaded withabout 60 μg cell lysate protein and electrophoresed at 100 V for 1-2 h.Proteins were then transferred from gels to Hybond-ECL™ membranes.Membranes were blocked in blocking solution (3% dry milk, 0.1% Tween 20in PBS) for 1 h at room temperature. Membranes were then incubated with1:1000 dilution of rabbit anti-human 3p peptides or anti-myc or FLAGmonocolonal antibodies, and 1:1000 dilution of mouse anti-β-actinmonocolonal antibodies. Immunocomplexes were detected with secondaryHRP-labeled rabit anti-mouse IgG or goat anti-rabit IgG antibodies usingan ECL™ kit (Amersham International).

EXAMPLE 8 Induction of Apoptosis by 3p Genes in Ad-3p-transduced TumorCells

[0314] The ability of exogenous 3p genes to induce apoptosis and theirimpact on cell-cycle processes in the Ad-3p-transduced H1299, A549,H460, H358, and HBEC cells were analyzed by FACS using the terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)reaction (FIG. 8). Induction of apoptosis was detected inAd-3p-transduced H1299 (FIG. 8A), A549 (FIG. 8B), and H460 (FIG. 8C)cells, but not in H358 (FIG. 8D) and HBEC (FIG. 8E) cells. More than15-20%, 40-65%, and 75% of cells were apoptotic at day 5 aftertransduction with Ad-101F6, Ad-Fus1, and Ad-Gene21 in the transducedH1299, A549, and H460 cells, respectively, whereas only about 7% and 10%of cells treated with PBS alone and transduced with Ad-EV vector,respectively, were TUNEL-positive at the same time periods. The level ofinduction of apoptosis in the Ad-3p-transduced cells increased with timeposttransduction and correlated with the viability of cells (FIG. 6).The inhibition of tumor cell proliferation by 3p genes are mediateddirectly or indirectly through induction of apoptosis.

EXAMPLE 9 Induction of Apoptosis and Alteration of Cell Cycle KineticsAd-3p-transduced Cells

[0315] Inhibition of tumor cell growth and proliferation by tumorsuppressor genes is usually characterized by induction of apoptosis andalteration of cell cycle processes. Thus, 3p gene-induced apoptosis andcell cycle kinetics were analyzed by flow cytometry using the terminaldeoxy transferase deoxyuridine triphosphate (dUTP) nick-end labelling(TUNEL) reaction with fluorescein isothiocyanate-labeled dUTP (Rochc,Molecular Biochemicals) and propidium iodide staining, respectively. Inbrief, cells (1×10⁶/well) are seeded on six-well plates and transducedwith Ad-3p constructs; untreated and Ad-EV-, Ad-GFP-, orAd-LacZ-transduced cells were used as controls. Cells were harvested atdesignated posttransduction times and then analyzed for DNAfragmentation and apoptosis by TUNEL reaction and for DNA content andcell cycle status by propidium iodide staining using flow cytometry,respectively, as described previously. The cell -cycle profiles in theAd-101F6, Ad-Fus1, and Ad-Gene21-transduced cells appeared to besignificantly affected by overexpression of these genes at later G2 andS phases stages compared to those in the untransduced andAd-EV-transduced controls at 3 days posttransduction (FIG. 9).

EXAMPLE 10 Suppression of Tumor Growth by Overexpression of 3p Genes InVivo

[0316] The tumor suppressor function of 3p genes, 101F6, Fus1, andGene2l were evaluated in vivo by direct intratumoral injection of theseAd-3p vectors into the A549 subcutaneous tumors in nude mice (FIG. 10).The growth of tumors was recorded from first injection until 20 daysafter last injection. All of the tumors in the mice treated withAd-101F6, Ad-Fus1, and Ad-Gene2l showed significantly suppressed growthcompared with tumors.

EXAMPLE 11. Effects of 3p Gene Expression on Tumorigenicity and TumorGrowth In Vivo

[0317] For the tumorigenicity study, H1299 or A549 cells were transducedin vitro with Ad-3p at an appropriate MOI with phosphate-buffered saline(PBS) alone as a mock control, Ad-EV as a negative control, and Ad-LacZas a nonspecific control. The transduced cells were harvested at 24 hand 48 h posttransduction, respectively. The viability of the cells wasdetermined by trypan blue exclusion staining. Viable cells (1×10⁷) werethen injected subcutaneously into the right flank of 6- to 8-week-oldfemale nude mice. Tumor formation in mice was observed two or threetimes weekly for up to 3 months. Tumor dimensions were measured every 2or 3 days.

[0318] To study the effect of 3p genes on tumor growth, H1299 or A549cells were used to establish subcutaneous tumors in nude mice. Briefly,1×10⁷ cells were injected into the right flank of 6- to 8-week-oldfemale nude mice. When the tumors reached 5 to 10 mm in diameter (atabout 2 weeks postinjection), the animals were intratumorally injectedwith Ad-3p and control vectors, respectively, 4 to 5 times within 10 to12 days for at a total dose of 3 to 5×10¹⁰ pfu per tumor. Tumor size wasmeasured and calculated as described above. At the end of theexperiment, the animals were killed and the tumors were excised andprocessed for pathological and immunohistochemical analysis.

EXAMPLE 12 Inhibition of Lung Metastatic Tumor Growth byProtamine-Adenovirus Complex-mediated 3p Gene Transfer In Vivo

[0319] The inventors have developed a novel Protamine/Adenovirus complexfor enhancement of the efficiency of adenovirus-mediated gene transferin vitro and for systemic delivery of recombinant adenovirus to lung andother organs in vivo by intravenous injection of the complex. TheProtemine-Ad-3p complexes (PAd3ps) were used to study the effects ofoverexpression of 3p genes on pulmonary metastatic tumor growth in A549experimental lung metastasis model in nude mice (FIG. 11). Themetastatic tumor growth was significantly inhibited in PAd-101F6,PAd-Fus1, and PAd-Gene21-treated mice, compared with those in controlgroups. These data are consistent with results obtained fromAd-3p-treated subcutaneous tumors. Therefore, the 3p genes play a rolein suppression of tumor growth and inhibition of tumor progression invivo.

EXAMPLE 13 Effect of 3p genes on metastatic tumor growth by LPD3p- orPAD3p-mediated 3p Genes Transfer In Vivo

[0320] The experimental lung metastasis models of H1299 and A549 cellswere used to study the effects of 3p genes on tumor progression andmetastasis by systematic treatment of lung metastatic tumors throughintravenous injection of either PAD3p or LPD3p complexes. A549 cells(1-2×10⁶) in 200 μl PBS were intra venially inoculated with nude miceand H1299 cells (1-2×10⁶) with SCID mice, respectively. Experimentalmetastatic tumor colonies were formed 7-10 days post-inoculation. PAD3psand control complexes were administered to animals by intravenousinjection every other two days for 3 times each at a dose of 2-5×10¹⁰viral particles/200-500 μg protamine, in a total volume of 200 μl peranimal. Alternatively, LPD3ps were applied by intravenous injectionevery day for 6 times each at a dose of 120 nmol liposome:6 μgprotamine:50 μg DNA, in a total volume of 200 μl per animal. Animalswere killed two weeks post last injection. Lung metastasis tumors werestained with Indian ink⁵¹, tumor colonies on the surfaces of lung werecounted under an anatomic microscope, and then the lung tissue weresectioned for further pathologic and immunohistochemical analysis.

EXAMPLE 14 Analysis of Telomerase Activity and Cellular Immortality

[0321] Activation of the enzyme telomerase, which has been associatedwith cellular immortality, may constitute a key step in the developmentof human cancer. Because of the nearly universal deregulated expressionof telomerase in lung cancer cells and the evidence for involvement of3p genes in the telomerase repression regulatory pathway, it will beimportant to study whether the alteration of tumor cell growth andproliferation implied by the introduction of wild-type 3p21.3 genes isassociated with repressed telomerase activity in Ad-3p transductants. Toassay telomerase activity, untransduced and Ad-3p- and controlvector-transduced cells (10⁵) are harvested and prepared as describedpreviously.³¹ The cell extract equivalent to approximately 10³, 10²,or10¹ cells is used for each telomerase assay. A standard telomeric repeatamplification protocol procedure, which is capable of detectingtelomerase activity in as few as 10 to 100 lung cancer cells, isperformed with modifications as described.^(32,45,46)

EXAMPLE 15. 123F2 (RASSF1A) in Lung and Breast Cancers and MalignantPhenotype Suppression³³⁹

[0322] I. Characterization of the 123F2 (RASSF1) Gene

[0323] To determine if the 123F2 (RASSF1A) gene was mutated in lung andbreast cancers, the inventors performed extensive mutational analysis ofthe RASSF1A isoform with the use of single-strand conformationpolymorphism assays on genomic DNA. The inventors had previously foundno RASSF1C mutations in 77 lung cancer cell line samples³⁴⁶. By use ofthe RASSFIA sequence as a reference, the inventors found severalpolymorphisms, including the following: codon 21 (AAG to CAG), Lys toGln; codon 28 (CGT to CGA), no amino acid change; codon 49 (GGC to GGT),no amino acid change; codon 53 (CGC to TGC), Arg to Cys; codon 129 (GACto GAG), Asp to Glu; codon 133 (GCT to TCT), Ala to Ser; and codon 325(TAT to TGT), Tyr to Cys. The 123F2 (RASSF1) gene is shown in FIG. 12.

[0324] II. Expression of RASSFIA and RASSF1C in Lung and Breast CancerCell Lines

[0325] 123F2 (RASSF1) is located within a region frequently affected byallele loss during growth of lung, breast, head and neck, kidney, andcervical tumors³⁴¹⁻³⁴⁵. The inventors investigated whether 123F2(RASSF1A) and RASSF1C are expressed in lung and breast cancer celllines. The inventors used isoform-specific RT-PCR to examine theexpression of 123F2 (RASSF1A) and RASSF1C in lung and breast tumor celllines and in normal lung and breast epithelial cultures (FIG. 13).

[0326] Isoform-specific RT-PCR assays were used for analysis of RASSF1Aand RASSF1C expression. Primers for RASSFIC were Nox3(5′-CTGCAGCCAAGAGGACTCGG-3′) and R182 and for RASSF1A were either PKCDFor NF (5′-TGCAAGTTCACCTGCCAC-3′) and R182 (FIG. 12. C). Total RNA wasisolated from previously described lung and breast cancer cell linesgrown in RPMI-1640 medium supplemented with 5% fetal bovine serum(complete medium) by Trizol extraction. Four micrograms of total RNA wasreverse transcribed by use of GIBCO-BRL Superscript First Strand cDNAKit. All cDNA preparations were tested for the ability to amplify anontranscribed genomic sequence immediately upstream of the first exonof the RASSF1A transcript. Any cDNAs that produced a product from thissequence were discarded because they were contaminated with genomic DNA.

[0327] The inventors also assessed the expression of RASSF1A afterexposure to 5-aza-2′-deoxycytidine, a drug that inhibits DNAmethylation. The inventors exposed subconfluent cultures of theRASSF1A-nonexpressing NSCLC line NCI-H157 to 0.5 p.M5-aza-2′-deoxycytidine for 48 hours, after which the inventors isolatedtotal RNA and performed RT-PCR for RASSF1A, RASSF1C, andglyceraldehyde-3-phosphate dehydrogenase (GAPDH). RT-PCR of GAPDHtranscripts was performed with the use of forward primer GAPDH-C(5′-CATGACAACTTTGGTATCGTG-3′) and reverse primer GAPDH-D(5′-GTGTCGCTGTTGAAGTCAGA-3′). RTPCR products were separated by agarosegel electrophoresis and visualized after staining with ethidium bromide.

[0328] 123F2 (RASSF1A) was expressed in normal lung epithelial cultures(NHBE and SAB cultures), in a normal breast epithelial culture (FIG. 13,C), but not in 32 (100%) of 32 SCLC lines, in 17 (65%) of 26 NSCLC celllines, and in 15 (60%) of 25 (60%) breast cancer cell lines.Representative data are shown in FIG. 13. By contrast, RASSF1C wasexpressed in nearly all of the lung and breast cancer cell lines tested,with the exceptions of several lung and breast cancer lines with knownhomozygous deletions that include the 123F2 (RASSF1) locus. In resectedlung adenocarcinomas, 123F2 (RASSF1A) was expressed in only two of fivecancers, while RASSF1C was expressed in all cancers (FIG. 13, C).

[0329] During RT-PCR analysis for 123F2 (RASSF1A), the inventorsfrequently noted two closely spaced bands in RASSF1A-expressing tumorsand in NHBE cultures (FIG. 13). The inventors sequenced these RT-PCRproducts and found that the larger band corresponded to 123F2 (RASSF1A),while the smaller product represented a different transcript, RASSF1F(GenBank Accession #AF286217). This transcript skips exon 1C to producean mRNA encoding a predicted truncated peptide of 92 amino acids, endingwithin the DAG-binding domain (FIG. 12. D). In nearly all of thesamples, RASSF1F is expressed when 123F2 (RASSF1A) is expressed.However, in some breast cancers and normal breast epithelial cultures(FIG. 13), 123F2 (RASSF1A) is expressed without RASSF1F expression.

[0330] III. Methylation Status of the 123F2 (RASSF1A) Promoter Region

[0331] Aberrant promoter methylation in tumors has been found to lead tothe loss of gene expression of several tumor suppressor genes in humancancers³⁴⁸. To assess whether the loss of 123F2 (RASSF1A) expression inlung cancer was the result of promoter hypermethylation, the inventorsdetermined the CpG methylation status in the 5′ region of 123F2(RASSF1A) (from −800 to +600 bp of the predicted 123F2 (RASSF1A)transcript start site) by sequencing sodium bisulfite-modified DNA fromeight lung cancer cell lines.

[0332] The methylation status of the presumed RASSF1A and RASSF1Cpromoter regions was determined by methylation-specific PCR. GenomicDNAs from lung cancer cell lines not expressing RASSF1A (NCI linesH1299, H1184, H1304, H841, H2108, and H128) or expressing RASSF1A (H1792and H2009) were modified by sodium bisulfite treatment^(352,353).Bisulfite treatment converts cytosine bases to uracil bases but has noeffect on methylcytosine bases. PCR amplification followed by sequencingof the PCR fragments identifies specific CpG dinucleotides in thepromoter region that are modified by methylation^(352,354,355). PCRprimers were designed to amplify genomic sequences in the presumedpromoter regions of RASSF1A (cosmid Lucal2; GenBank Accession #AC002481nucleotides 17730-18370) and RASSF1C (GenBank Accession #AC002481nucleotides 21022-21152 and 21194-21332). The resulting PCR fragmentswere sequenced by automated fluorescence-based DNA sequencing todetermine the methylation status.

[0333] The data on CpG methylation in RASSF1A-nonexpressing lung cancercell lines were used to design methylation-specific PCR³⁵² primers forthe RASSF1A 5′ promoter region: The primers to detect the methylatedform were 5′-GGGTTTTGCGAGAGCGCG-3′ (forward) and5′-GCTAACAAACGCGAACCG-3′ (reverse), and the primers to detect theunmethylated form were 5′-GGTTTTGTGAGAGTGTGTTTAG-3′ (forward) and5′-CACTAACAAACACAAACCAAAC-3′ (reverse). Each primer set generated a169-basepair (bp) product. Methylation-specific PCR cycling conditionsconsisted of one incubation of 15 minutes at 95° C., followed by 40cycles of a 30-second denaturation at 94° C., 50 seconds at an annealingtemperature (64° C. for methylation-specific and 59° C. forunmethylated-specific primers), a 30-second extension at 72° C., and afinal extension at 72° C. for 10 minutes. PCR products were separated in2% agarose gels. Lymphocyte DNA, methylated in vitro by CpG (SssI)methylase (New Engiand Biolabs, Inc., Beverly, Mass.) following themanufacturer's directions, was used as a positive control. A water blankwas used as a negative control.

[0334] All of the six lung cancer cell lines not expressing 123F2(RASSF1A) exhibited methylation of almost all CpG dinucleotide sites inthe putative promoter region. The two lung cancer cell lines that didexpress 123F2 (RASSF1A either were not methylated at these CpG sites orshowed limited methylation. By contrast, no methylation was found in CpGsites in the presumed RASSF1C promoter region of these eight cell lines.

[0335] To confirm that promoter hypermethylation contributes to the lackof expression of 123F2 (RASSF1A) in the lung cancer cell lines, theinventors assessed the effect of 5-aza-2′-deoxycytidine, a drug thatinhibits DNA methylase, on 123F2 (RASSF1A) expression. The inventorsexposed the RASSF1A-nonexpressing NSCLC line NCI-H157 to5-aza-2′-deoxycytidine and found re-expression of 123F2 (RASSF1A) bythis cell line but little or no change in the expression of thehousekeeping gene GAPDH or in the expression of RASSF1C (FIG. 14).

[0336] IV. Methylation-Specific PCR Analysis of the Promoter Region of123F2 (RASSF1A) in Lung and Breast Cancers

[0337] To determine the methylation status of the promoter region ofRASSF1A in primary lung and breast cancers, the inventors usedmethylation-specific PCR analysis. Genomic DNA from a large number ofprimary resected NSCLCs, paired lung tissues resected from the samepatients but not involved with the cancer, primary resected breastcancers, and a large panel of lung and breast cancer cell lines weretreated with sodium bisulfite and tested for the presence of methylatedand unmethylated CpG dinucleotides in the promoter region of 123F2(RASSF1A) (FIG. 15). All of the primary resected NSCLCs andnon-tumor-paired samples contained unmethylated promoter sequences,which were expected because these resected tumors were notmicrodissected and were contaminated with stromal cells. However, 32(30%) of 107 primary NSCLCs, 47 (100%) of 47 SCLC lines, and 19(49%) of39 primary breast cancers exhibited the methylated RASSFIA allele (FIG.15; Table 7). By contrast, no methylated alleles were detected in 104paired resected nonmalignant lung tissues (FIG. 15; Table 7). TABLE 7Frequency of methylation-specific polymerase chain reaction assay fordetection of RASSF1A CpU island-methylated alleles in lung and breastcancers No. of methylation DNA sample source* No. tested alleles(positive) (%) Primary resected NSCLCs 107 32 (30%) Correspondingnonmalignant lung 104 0 (0%) NSCLC lines  27 17 (63%) SCLC lines  47  47(100%) Primary resected breast cancers  39 19 (49%) Breast cancer lines 22 14 (64%)

[0338] The inventors found a high frequency of methylated 123F2(RASSF1A) alleles in the panel of lung and breast cell cancer lines(Table 7). Because the lung and breast cancer cell lines representessentially clonal populations of cancer cells without contaminatingnormal cells. the inventors tabulated the frequency of the methylatedand unmethylated 123F2 (RASSF1A) alleles (Table 8). While the lung andbreast cancer lines often derive from clinically more aggressive lesionsthan the average population of tumors³⁴⁹⁻³⁵¹, the inventors previousstudies^(350,351) have shown that cancer cell lines continue to retainthe genetic alterations found in the uncultured cancer specimens fromwhich they were derived. The presence of only the methylated allele isconsistent with either the methylation of both parental alleles or theretention of the methylated allele and the loss of the unmethylated 3pallele. All of the SCLC cell lines howed only the methylated allele orlacked 123F2 (RASSF1A) entirely because of a homozygous deletion,consistent with the nearly universal 3p21.3 allele loss inSCLC^(341,350,356). Of the NSCLC cell lines, 13 (48%) of 27 (Table 8)had only the methylated 123F2 (RASSF1A) allele, and 10 (37%) of 27 hadonly the unmethylated allele, consistent with a lower rate of 3p21.3allele loss in this tumor type³⁴¹. Likewise, 10 (45%) of 22 samples(Table 8) of breast cancer cell lines had only the methylated allele,and seven (32%) of 22 had only the unmethylated allele, again consistentwith the rate of 3p21.3 allele loss found in breast cancer³⁵¹. Asexpected, two tumor lines shown previously to have homozygous deletionsinvolving the 3p21.3 region were negative for both the methylated andthe unmethylated allele (Table 8)^(346,347). TABLE 8 Presence ofmethylated and unmethylated RASSF1A alleles in 97 lung and breast cancercell lines* RASSF1A CpG genotype Methylated Unmethylated allele alleleSCLC NSCLC BCCL Total + + 0 4 4 8 + − 47  13  10  70  − + 0 10  7 17  −− 1 0 1 2^(†) Total 48  27  22  97 

[0339] For a subset of 61 lung and breast cancer cell lines, theinventors performed both expression and methylation analysis and found astatistically significant association (P<0,001, Fisher's exact test)between the presence of methylated RASSF1A alleles and the loss of 123F2(RASSF1A) expression. In 12 samples, 123F2 (RASSF1A) was expressed inthe absence of a methylated allele; in 44 samples, 123F2 (RASSF1A) wasnot expressed in the presence of a methylated allele; in four samples,123F2 (RASSF1A) was not expressed in the absence of methylated allele;and in one sample (a breast cancer cell line), 123F2 (RASSF1A) wasexpressed in the presence of both a methylated and an unmethylatedallele. These data show the critical association of 123F2 (RASSF1A)methylation with loss of 123F2 (RASSF1A) expression.

[0340] The inventors next assessed whether there was any associationbetween 123F2 (RASSF1A) promoter methylation and clinical findings inthe patients with primary NSCLC. The inventors found no statisticallysignificant association between 123F2 (RASSF1A) methylation and age,sex, tumor-node-metastasis (TNM) pathologic stage, or tumor histology in107 resected NSCLCs. In addition, the inventors found no statisticallysignificant association between 123F2 (RASSF1A) methylation and age, TNMpathologic stage, tumor histology, estrogen or progesterone receptorstatus, or HER2/Neu expression in 39 primary resected breast cancers.

[0341] Survival among lung cancer patients differed by the methylationstatus of 123F2 (RASSF1A) (P=0.046) (FIG. 16). Also, by univariateanalysis, in this group of 107 patients with NSCLC treated with anattempt at curative surgical resection, tumor (T1, T2, and T3), lymphnode stage (N1 and N2), and reported weight loss were statisticallysignificant predictors of adverse survival. Neither smoking history(yes/no or pack-years with 40 pack-year cutoff) nor treatmentdifferences (all patients had surgical resection of lobectomy orpneumonectomy, and only five had prior radiotherapy or chemotherapy)accounted for the adverse survival. Because a multivariate analysis isof limited use with a small sample size, the inventors performed a Coxproportional hazards regression analysis by use of 123F2 (RASSF1A)methylation and the main univariate factors (tumor, lymph node stage,and weight loss). 123F2 (RASSF1A) methylation was not found to be anindependent prognostic factor of survival. However, this result could bedue to small numbers because even lymph node stage (a known prognosticfactor) was also no longer an independent factor in the analysis.

[0342] V. Effect of Exogenous Expression of 123F2 (RASSF1A) on TumorCell Phenotype

[0343] The inventors examined the effect of RASSF1A on the tumor cellphenotype by three methods. The inventors used anchorage-dependentcolony formation as a measure of proliferation and anchorage-independentcolony formation as a measure of malignant potential. The inventors alsodirectly assessed in vivo tumor formation.

[0344] The in vitro growth characteristics of NSCLC NCI-H 1299 clonesthat express RASSF1A were tested for anchorage-dependent andanchorage-independent (soft agar) growth. After 48 hours of growth innonselective medium, transiently transfected NSCLC NCI-H 1299 cells weredetached with trypsin and diluted, usually 10-to 25-fold, incomplete-medium containing 800 μg/mL of G418 and plated into fresh100-mm dishes. The medium was changed twice weekly. After 14 days, themedium was removed, the plates were washed with phosphate-bufferedsaline (PBS), and the colonies were stained with 1% methylene blue in50% (vol/vol) ethanol. For the anchorage-independent, soft agar-growthassays, 1000 RASSFIA-expressing cells were suspended and plated in 0.33%Noble agar (Sigma Chemical Co.. St. Louis, Mo.) in complete mediumsupplemented with 600 μg/mL G418 and layered over a 0.50% agar base incomplete medium. After 21 days, colonies greater than 0.2 mm in diameterwere counted.

[0345] For retrovirally infected cells, anchorage-independent growthassays were performed as follows: 10000 viable selected cells from eachinfection were plated in 0.33% soft agar over a 0.50% agar base inDulbecco's modified Eagle medium (Life Technologies, Inc.) with 10%heat-inactivated fetal bovine serum. After 21 days, colonies greaterthan 0.2 mm in diameter were counted.

[0346] The inventors also tested the ability of RASSF1A-infected cellsto grow in vivo in nude mice. Male BALB/c nude (nu/nu) 3- to 6-week-oldmice were irradiated on day 0 of the experiment in groups of fiveaaimals by a 5-minute exposure to 350 cGy from a cesium source. The nextday, each mouse was given an injection subcutaneously on its flank with0.2 mL of sterile PBS containing 107 viable parental, vector control, orRASSF1A retroviral-infected NSCLC NCI-H1299 tumor cells. Mice weremonitored every 2-3 days for tumor size; once tumors reached greaterthan 1500 mm³, the mice were killed.

[0347] The inventors first cloned RASSF1A cDNA into pcDNA3.1+, anexpression vector that contains a selectable marker, and transfectedNCI-H1299 cells, which lack endogenous 123F2 (RASSF1A) expression. Afterselection for 14-21 days, the inventors determined colony formation ofNCI-H1299 cells in both anchorage-dependent and anchorage-independentassays. Expression of 123F2 (RASSF1A) in NCI-H 1299 cells resulted in a40%-60% decrease in anchorage-dependent colony formation and in anapproximate 90% decrease in anchorage-independent colony formationcompared with cells transfected with the pcDNA3. 1 vector alone (FIG.17, A). Because NCI-H1299 cells have an intragenic p53 homozygousdeletion, transient expression of wild-type p53 can serve as a positivecontrol for growth inhibition. Indeed, expression of wild-type p53 inNCI-H 1299 cells resulted in a 80% and 95% reduction in colony formationin anchorage-dependent and anchorage-independent assays, respectively(FIG. 17, A). Several clones of NCI-H1299 cells transfected with 123F2(RASSF1A) were isolated in selective medium and were found to express123F2 (RASSF1A) by northern blot analysis (FIG. 17, B). Although theclones grew well in vitro, each had reduced anchorage-independent colonyformation by approximately 90% compared with the vector-transfectedcontrol clones (FIG. 17, C).

[0348] To eliminate the possibility that the pcDNA3.1+ vector mediatedthe growth-suppression effects, the inventors infected NCI-H 1299 cellswith retroviral-expression vectors containing 123F2 (RASSF1A) or RASSF1Cand tested the ability of these cells to grow in ananchorage-independent manner. Cells expressing 123F2 (RASSF1A) had amarked reduction in the ability to form soft-agar colonies compared withcells infected with the retroviral empty vector or the retroviral vectorcontaining RASSF1C (FIG. 17, D). Cells expressing the retroviral vectorformed 3200 colonies per 10000 cells plated. 123F2 (RASSF1A)-expressingcells formed only 19% of the vector control colonies, while RASSF1Cformed 108% of the vector control. RASSF1A- and RASSF1C-infected cellsgrew well in vitro and showed no signs of toxicity or apoptosis.

[0349] Finally, the inventors tested the ability of the retrovirallyinfected NCIH1299 cells to form tumors in nude mice. Cells transfectedwith the vector (parental cells) formed tumors rapidly (FIG. 17, E). Bycontrast, cells infected with 123F2 (RASSF1A) retroviral vector andexpressing the 123F2 (RASSF1A) protein had much lower tumorigenicity invivo (FIG. 17, E).

EXAMPLE 16 Several Genes in the Human Chromosome 3p21.3 HomozygousDeletion Region Exhibit Tumor Suppressor Activities in vitro and in vivo

[0350] I. Effects of forced expression of 3p genes on Tumor Cell Growth.

[0351] To test the hypothesis that one or more of the 3p genes functionas tumor suppressors in vitro, the inventors performed a series ofexperiments to study the effects of expression of the 3p21.3 genes oncell proliferation in several types of Ad-3p-transduced human NSCLCcells and a normal HBEC line. Cells in each line were transduced invitro by Ad-10F6, Ad-FUS1, Ad-NPRL2, Ad-BLU, Ad-RASSF1, Ad-HYAL2 andAd-HYAL1 vectors at various MOIs in units of vp/c; cells were treatedwith PBS, Ad-EV, Ad-LacZ, or Ad-p53 as mock, negative, non-specific, orpositive controls, respectively. The transduction efficiency wasdetermined by examining GFP-expressing cells in the Ad-GFP-transducedcell population under a fluorescence microscope and was found to begreater than 80% at the highest MOI applied for each cell line.

[0352] Cell proliferation was analyzed by using the XTT assay todetermine the number of viable cells remained at 1, 2, 3, and 5 daysafter transduction {only data for day 5 at highest MOIs (5000 vp/c forA549, 1000 vp/c for H1299, 5000 vp/c for H460, 2500 vp/c for H358, and1000 vp/c for HBE, respectively) are shown} (FIG. 19). In all cases, theviability of transduced cells was compared with that of untransduced(PBS-treated) control cells (whose viability was set at 100%). As can beseen in FIG. 22, cell viability was significantly reduced in Ad-101F6-,Ad-Fus1-, and Ad-NPRL2-transduced A549 and H460 cells, which showhomozygousity for multiple 3p21.3 markers and contain wild-type p53, andH1299 cells, which exhibit 3p21.3 homozygous but also have a homozygousdeletion of p53. A modest reduction of cell viability was shown inAd-RASSF1C-transduced H1299 cells. However, no significant effect ongrowth was observed in any of these cells transduced with Ad-HYAL1,Ad-HYAL2, Ad-BLU, Ad-EV or Ad-LacZ. These results suggest that exogenousexpression of some wild-type 3p21.3 genes could inhibit 3p-deficienttumor cell growth or restore the tumor suppressor function of these3p21.3 genes in vitro.

[0353] To clarify the specificity of the observed inhibitory effects ontumor cell growth and examine the potential cytotoxicity of theexogenously expressed 3p21.3 genes on normal cells, the inventorsanalyzed the effects of these 3p21.3 genes on cell proliferation in3p21.3 heterozygous H358 cells and normal HBECs (FIG. 19). As shown inFIG. 19, HBECs transduced with all Ad-3p genes at highest MOIs hadlosses of cell viability of less than 10%, while H358 cells transducedwith the same vectors had losses of cell viability less than 20% whencompared with the untransduced control cells. Similar levels of lossesof cell numbers were observed in H358 and HBEC cells transduced withAd-EV and Ad-LacZ. H358 cells which are deleted for p53 showed reducedcell viability when transduced with the Ad-p53 control. These resultscouple with the lack of effect with Ad-LacZ, Ad-HYAL2, Ad-HYAL1,Ad-RASSF1, and Ad-BLU, demonstrate the specificity of thetumor-suppressing function of 3p21.3 genes, FUS1, NPRL2, 101F6 in3p-deficient tumor cells and indicate that no generalized cytotoxicitywas associated with exogenous expression of these wild-type 3p21.3genes.

[0354] Expression of 3p21.3 genes in Ad-3p transfectants was verified byquantitative real-time RT-PCR, and known concentrations of human totalRNA and primers and TaqMan probe for β-actin DNA and for GAPDH cDNA wereused as standards and internal controls, respectively (FIG. 20). Thetranscription of FUS1 (FIG. 20A), 101F6 (FIG. 20B), NPRL2 (FIG. 20C),and HYAL1 (FIG. 20D) was demonstrated quantitatively by showing theassociation between increased levels of expression of these 3p21.3 geneswith increased MOIs of the corresponding Ad-3p vectors in transducedH1299 cells. The transcription of other 3p21.3 genes, HYAL2, HYAL1, BLU,and RASSF1, was also detected by real-time RT-PCR. The expression ofFUS1 and 101F6 proteins was detected also by western blot analysis usingavailable polyclonal antibodies raised against the oligopeptides derivedfrom their deduced amino acid sequences.

[0355] II. Induction of Apoptosis by 3p Genes in Ad-3p-transduced TumorCells.

[0356] The ability of exogenously expressed 3p21.3 genes to induceapoptosis in the Ad-3p-transduced H1299, A549, H460, H358, and HBECcells was analyzed by FACS using the TUNEL reaction (FIG. 21). Inductionof apoptosis was detected in Ad-101F6-, Ad-FUS1-, andAd-NPRL2-transduced A549 (FIG. 21A), H1299 (FIG. 21B), and H460 (FIG.21C) cells, but not in H358 (FIG. 21D) and HBEC (FIG. 21E) cells. Theapoptotic cell populations increased with increased duration oftransduction; more than 15-20%, 40-65%, and 75% of cells were apoptotic5 days after transduction with Ad-101F6, Ad-FUS1, and Ad-NPRL2 in thetransduced H1299, A549, and H460 cells, respectively, whereas only about7% and 10% of cells treated with PBS alone and transduced with Ad-EVvector, respectively, were TUNEL-positive after the same time interval.The levels of apoptosis induction by Ad-101F6, Ad-FUS1, and Ad-NPRL2appeared 20-50% more significant in A549 and H460 cell lines withwild-type p53 genes (FIG. 21A and 21C) than that in H1299 cell linedeleted for p53 gene (FIG. 21B). Levels of apoptosis in A549 and H4(50cells were comparable to those induced by Ad-p53 in p53-deficient H1299and H358 cells (FIG. 21B and D). However, no significant induction ofapoptosis was observed in any tumor cell line transduced by Ad-BLU,Ad-RASSF1, Ad-HYAL2, and Ad-HYAL1 (FIG. 21). The levels and time ofinduction of apoptosis in cells transduced by these Ad-3p vectors werewell correlated with those of cell proliferation inhibition in cellstreated with the same vectors (FIG. 19), suggesting that suppression oftumor cell proliferation by 3p21.3 genes is mediated directly orindirectly through a mechanism of apoptosis induction.

[0357] III. Suppression of Tumor Growth by Intratumoral Injection ofAd-3p Vectors.

[0358] To determine whether the observed inhibitory effects of these3p21.3 genes on tumor cell proliferation in vitro could be demonstratedon tumor growth in vivo, the inventors evaluated the efficacy of 3p21.3genes in suppressing tumor growth by direct intratumoral injection ofAd-3p21.3 gene vectors, along with PBS and Ad-EV, Ad-LacZ, and Ad-p53vectors as controls, into A549 or H1299 tumor xenografts in nu/nu mice(FIG. 22). The growth of tumors was recorded from the first injectionuntil 20 days after the last injection. Tumor volumes were normalized bycalculating the percentage increase in tumor volume after treatmentrelative to volume at the beginning of treatment in each group. In bothA549 (FIG. 22A) and H1299 (FIG. 22B) tumor models, all of the tumorstreated with Ad-101F6, Ad-FUS1, or Ad-NPRL2 showed significantlysuppressed growth (P<0.001), compared with mouse groups treated withAd-LacZ or Ad-EV controls, whereas no significant effect was observed inAd-BLU, Ad-RASSF1, and Ad-HYAL1-treated tumors. H1299 A549 tumorxenografts but not A549 H1299 tumors treated with Ad-HYAL2 showedsignificant reduction only at the end points of treatment (P=0.036).Moreover, a significantly stronger inhibition of tumor growth was shownin A549 tumors treated with Ad-101F6 and Ad-NPRL2 vectors than in tumorstreated with the Ad-p53 vector (FIG. 22A).

[0359] IV. Inhibition of Development of Experimental Lung Metastases byProtamine-Adenovirus Complex-mediated 3p21.3 Gene Transfer.

[0360] A novel formulation using protamine/adenovirus complexes(designated P-Ad) for enhanced systemic delivery of recombinantadenovirus in vivo was developed to further explore the potential of3p21.3 genes in suppressing systemic metastases. An experimental A549metastatic human lung cancer model was used to study the effects of3p21.3 gene transfer on the development of lung metastases in nu/nu mice(FIG. 23). The adenoviral 3p21.3 gene vectors were complexed toprotamine and delivered via intravenous injection. The development ofA549 metastases was significantly inhibited and the formation ofmetastatic tumor colonies on the surfaces of lungs from mice inoculatedwith A549 was reduced more than 80% in animals treated with P-Ad-101F6,P-Ad-FUS1, P-Ad-NPRL2, P-Ad-BLU or P-Ad-HYAL2 compared with those incontrol treatment groups (FIG. 23A). However, no significant reductionof metastatic colony formation was observed in animals treated withP-Ad-HYAL1 and P-Ad-RASSF1P-Ad-BLU. These data are consistent withresults obtained from Ad-3p-treated subcutaneous tumors, furthersupporting the roles of these 3p21.3 genes in suppression of tumorgrowth and inhibition of tumor progression in vivo.

EXAMPLE 17 Overexpression of candidate tumor suppressor gene FUS1isolated from the 3p 21.3 homozygous deletion region leads to G1 arrestand growth inhibition of lung cancer cells

[0361] Very frequent loss of one allele of chromosome arm 3p in bothsmall lung cancer (SCLC) and non-small cell lung cancer (NSCLC) providesstrong evidence for the existence of tumor suppressor genes (TSGs) inthis chromosome region^(363;364;367;371;372). Multiple different 3pregions showing isolated allele loss were identified by detailedallelotyping studies suggesting there are several different TSGs locatedon 3p suggesting there are several different TSGs located on3p^(361;362;372). Nested homozygous deletions in lung cancer and breastcancer cell lines have been found at 3p21.3 that focused our search on a630 kb region including the identification, annotation, and evaluationof 25 new genes as TSG candidates^(357;365;366;368;369;370). A breastcancer deletion narrowed this region further to 120 kb and 9 TSGcandidates (CACNA2D2, PL6, 106F6, NPRL2/g21, BLU, RASSF1, FUS1, HYAL2,HYAL1) were located in or bordering this region³⁶⁹. One of thesecandidate TSGs, FUS (AF055479), did not show homology with any knowngenes, was found to have only few mutations in lung cancers, and usuallywas expressed at the mRNA level in lung cancers³⁶⁶. Several NSCLCs(NCI-H322 and NCI-H1334) exhibited the same nonsense mutation, whicharose from aberrant mRNA splicing. This aberrant form lacked 28 bp ofmRNA at the 3′ terminus of FUS1 exon 2 resulting in a truncatedpredicted protein of 82 amino acids compared to 110 amino acids in thewild-type (FIG. 24). To confirm the inventors mutational analysis, whichpreviously had been conducted on lung cancer cell line DNAs, theysearched for other mutations in FUS1 in primary uncultured lung cancers.Single strand conformation polymorphism (SSCP) analysis was performedusing genomic DNA of 40 primary uncultured lung cancers (9 SCLCs and 31NSCLCs) (FIG. 24)³⁶⁰. No mutations were detected although the inventorsfound a single nucleotide polymorphism in intron 2 that did not alterthe amino acid sequence of FUS1.

[0362] The inventors next considered CpG island promoter regionmethylation as an epigenetic mechanism leading to TSG inactivation. Infact, such tumor acquired promoter region methylation was found to occurfor the RASSF1A mRNA isoform residing immediately centromeric to FUS1^(354; 358). However, FUS1 mRNA was expressed in most lung cancersmaking such CpG methylation an unlikely method of inactivation ofFUS1³⁶⁶. In addition, the 5′ putative promoter region containing CpGislands of FUS1 was sequenced using sodium bisulfite treated³⁵⁵ DNA from6 lung cancers were the inventors did not detect FUS1 protein expressionand found no CpG methylation.

[0363] The possibility of haploinsufficiency or reduced expression ofFUS1 was considered as another mechanism for this gene to participate inlung cancer pathogenesis^(356; 359; 373). The inventors first performedwestern blot analysis of a panel of lung cancer cell lines using ananti-Fus1 anti peptide antibody which readily detected exogenouslyexpressed Fus1 (FIG. 25) but could not detect any endogenous FUS1expression in lung cancers (FIG. 25 for H1299 NSCLC given as an exampleof negative data). This lack of detection could be due to a variety offactors including the quality of the antibodies. Nevertheless, if lossor low levels of FUS1 protein expression was involved in lung cancerpathogenesis the inventors reasoned that exogenous introduction andexpression of Fus1 might suppress the malignant phenotype. Colonyformation assays were performed after transfection of FUS1 expressionvectors. The inventors made a C terminal FLAG-tagged FUS1 construct byPCR and ligated it into expression vector pcDNA3.1 (Invitrogen, CarlsbadCalif.). Empty vector and an expression vector containing wild-typeFUS1, FLAG-tagged FUS1, and the 82 aa mutant FUS1 werewas transfectedinto NSCLC NCI-H1299 cells which has suffered allele loss for the 3p21.3630 kb region and does not express detectable FUS1 protein (FIG. 25),and NSCLC NCI-H322 cells containing a expressing theendogenoushomozygous nonsense truncation mutation of FUS1 1 and also notexpressing detectable FUS1 protein. Expression of the FUS1 constructs inH1299 cells after transient transfection was confirmed by Western blotanalysis using anti-Flag and anti-N terminal FUS1 antibodies (FIG. 25).The effect of FUS1 transfection with a neo resistance gene on lungcancer colony formation was tested. The numbers of G418 resistantcolonies in the FUS1 transfections were dramatically reduced incomparison with transfection with the empty vector (FIG. 25). Bycontrast, the number of colonies formed in the mutant FUS1 transfectantswas only slightly reduced, suggesting that this lung cancer-associatedmutant FUS1 was functionally inactive (FIG. 25).

[0364] An ecdysone inducible mammalian expression system in H1299 cellswas developed to confirm that overexpression of FUS1 could inhibit tumorcell growth. In this system, FUS1 expression is induced in the presenceof Ponasterone A. H1299 parent ECR9 cells with the regulatable hormonereceptor vector pVgRXR alone served as an additional control. H1299 ECR9cells were transfected with pINDsp1-FUS1-FLAG(neo), selected with G418in the presence or absence of Ponasterone A, and compared the numbers ofG418 resistant colonies. The number of colonies formed in cells withinduced expression of in the FUS1 induced condition was decreased anaverage of 75±8% compared with number of colonies in cells undertheuninduced condition, providing another confirming ation of the growthinhibitory activity of FUS1. Twenty stable G418 resistant clones wereisolated in the uninduced condition and , the inducible expression ofFUS1-FLAG was examined., Among them, 6 clones showed some FUS1 inductionand two stable clones were selected (Cl.13 and Cl.16) in whichexpression of FUS1-Flag wasas clearly indicible by Ponasterone A (FIG.26). However, both cell lines expressed some FUS1 in the uninducedcondition, indicating that regulation of FUS1 expression was leaky.

[0365] The cell growth rate was examined in induced and uninducedconditions by the MTT assay. Ponasterone A has no effect on the growthof parental cell line H1299 ECR 9 cells, but the growth of Cl. 13 andCl. 16 cells were inhibited in the presence of Ponasterone A (FIG. 26).The induction of Fus1 expression and inhibition of tumor cell growthappeared to be dependent on the dose of Ponasterone A both increasingwith the increased concentrations of Ponasterone A (FIG. 26). With Fus1induction, the doubling times of the tumor cells were also increased inboth clones, from 22 to 46 hrs for Cl. 13 and from 21 to 45 hrs forCl.16, respectively. These results also indicated that overexpression ofFus1 suppresses H1299 lung cancer cell growth in vitro.

[0366] An increase of apoptosis in H1299 cells under induced conditionby TUNEL assay was not observed. However, when cells were induced byPonasterone A to express Fus1 for 48 hrs and analyzed by fluorscentactivated cell sorter (FACS) analysis (see legend of FIG. 26 fordetails) by FACS analysis the inventors found: parental H1299-ECR9 cellsto have unchanged cell cycle parameters (G1 51%, S 18%, G2/M 31%uninduced and G1 50%, S 18%, G2/M 32% induced); while Fus1 inducedclones showed G1 arrest (H1299 clone13 showed G1 50%, S 17%, G2/M 33%uninduced and G1 65%, S 10%, G2/M 25% induced; and H1299 clone16 G1 56%,S 16%, G2/M 28% uninduced and G1 65%, S 12%, G2/M 23% induced). Theincrease in G1% was significant (P<0.05, students t test). These resultssuggest of cell cycle analysis showedthat overexpression of FUS1 inH1299 cells is associated with G1 arrest and alteration of cell cyclekinetics.

[0367] Lung cancer cell lines do not express detectable endogenouslevels of Fus1 protein, and exogenous introduction of Fus1 withoverexpression inhibited lung cancer cell growth in vitro. This growthinhibition was seen in a lung cancer line suffering allele loss for theregion and in another carrying a homozygous truncating mutation of FUS1.In addition, the inventors found that this truncated Fus1 protein hadlost tumor growth suppressing activity. Besides the acute transfectionstudies, the inventors established a Fus1 inducible system and showedthat tumor growth inhibition was correlated with the level of expressionof Fus1 protein. In addition, cell cycle analysis using the sameexpression- regulatable system showed that the mechanism for theinhibition of cell growth was associated with G1 arrest and not withinduction of apoptosis. Finally, the inventors confirmed that somaticmutation of FUS1 was rare in primary lung cancers (0/40), in agreementwith previous studies which showed 3/79 lung cancers with alterations inthe FUS1 gene (2 nonsense mutations and 1 deletion). In fact thefrequency of mutation in any of the 22 out of 25 candidate genes theinventors have studied in detail in this 600 kb 3p21.3 region is lowcompared to the high frequency LOH at this locus. One possibility toaccount for the low mutation frequency is loss of expression of FUS1 orother of the 3p21.3 genes by tumor promoter acquired methylation. Theexpression of RASSF1A mRNA isoform isolated from the same 3p21.3deletion region and 15.5 kb centromeric of FUS1 was repressed in manylung cancers by acquired CpG island promoter DNA methylation for thisgene^(354; 358). Replacement of RASSF1A inhibited tumor cell growth invitro and in vivo indicating RASSF1A is another candidate tumorsuppresser gene in this locus. However the inventors have not found lossof FUS1 mRNA expression³⁶⁶ or 5′ region CpG methylation for FUS1 in lungcancers thus excluding tumor acquired promoter methylation as aninactivating mechanism for the FUS1 gene. FUS1 may act ashaploinsufficient tumor suppressor gene³⁵⁶. The inventors experimentsshowed that overexpression of FUS1 caused G1 arrest in H1299. Althoughsome signal or environmental cue may induce the expression of Fus1 andlead to G1 arrest in normal cells, 3p allelic loss and some otheralteration of FUS1 in malignant cells may lead to haploinsufficiencyand/or loss of expression of FUS1 in lung tumors and escape from cellcycle arrest.

[0368] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

References

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1 2 1 1746 DNA Homo sapiens 1 gatccccaac cgtcccgcaa ctgtcctgtcccagactttg gcaccgtcgg ggtccgtcgt 60 ccccgaatgt gacagcatcc ccaccccggctgctgcccag gatccgccgg accccggcct 120 cgatatggga gacctggaac tgctgctgcccggggaagct gaagtgctgg tgcggggtct 180 gcgcagcttc ccgctacgcg agatgggctccgaagggtgg aaccagcagc atgagaacct 240 ggagaagctg aacatgcaag ccatcctcgatgccacagtc agccagggcg agcccattca 300 ggagctgctg gtcacccatg ggaaggtcccaacactggtg gaggagctga tcgcagtgga 360 gatgtggaag cagaaggtgt tccctgtgttctgcagggtg gaggacttca agccccagaa 420 caccttcccc atctacatgg tggtgcaccacgaggcctcc atcatcaacc tcttggagac 480 agtgttcttc cacaaggagg tgtgtgagtcagcagaagac actgtcttgg acttggtaga 540 ctattgccac cgcaaactga ccctgctggtggcccagagt ggctgtggtg gcccccctga 600 gggggaggga tcccaggaca gcaaccccatgcaggagctg cagaagcagg cagagctgat 660 ggaatttgag attgcactga aggccctctcagtactacgc tacatcacag actgtgtgga 720 cagcctctct ctcagcacct tgagccgtatgcttagcaca cacaacctgc cctgcctcct 780 ggtggaactg ctggagcata gtccctggagccggcgggaa ggaggcaagc tgcagcagtt 840 cgagggcagc cgttggcata ctgtggccccctcagagcag caaaagctga gcaagttgga 900 cgggcaagtg tggatcgccc tgtacaacctgctgctaagc cctgaggctc aggcgcgcta 960 ctgcctcaca agttttgcca agggacggctactcaagctt cgggccttcc tcacagacac 1020 actgctggac cagctgccca acctggcccacttgcagagt ttcctggccc atctgaccct 1080 aactgaaacc cagcctccta agaaggacctggtgttggaa cagatcccag aaatctggga 1140 gcggctggag cgagaaaaca gaggcaagtggcaggcaatt gccaagcacc agctccagca 1200 tgtgttcagc ccctcagagc aggacctgtggctgcaggcg cgaaggtggg ctgagaccta 1260 caggctggat gtgctagagg cagtggctccagagcggccc cgctgtgctt actgcagtgc 1320 agaggcttct aagcgctgct cacgatgccagaatgagtgg tattgctgca gggagtgcca 1380 agtcaagcac tgggaaaagc atggaaagacttgtgtcctg gcagcccagg gtgacagagc 1440 caaatgaggg ctgcagttgc tgagggccgaccacccatgc caagggaatc cacccagaat 1500 gcacccctga acctcaagat cacggtccagcctctgccgg agccccagtc tccgcagtgg 1560 agagcagagc gggcggtaaa gctgctgaccgatctccctc ctcctcaccc caagtgaagg 1620 ctcgagactt cctgccccac ccagtgggtaggccaagtgt gttgcttcag caaaccggac 1680 caggagggcc agggccggat gtggggaccctcttcctcta gcacagtaaa gctggcctcc 1740 agatcg 1746 2 362 PRT Homo sapiens2 Met Gln His Pro His Pro Gly Cys Cys Pro Lys Pro Arg Pro Arg Tyr 1 5 1015 Gly Arg Pro Gly Thr Ala Ala Ala Arg Gly Ser Ser Ala Gly Ala Gly 20 2530 Ser Ala Gln Leu Pro Ala Thr Arg Arg Val Glu Pro Ala Ala Glu Pro 35 4045 Gly Glu Ala His Pro Arg Cys His Ser Gln Pro Gly Arg Ala His Ser 50 5560 Gly Ala Ala Gly His Pro Trp Glu Gly Pro Asn Thr Gly Gly Gly Ala 65 7075 80 Asp Val Glu Ala Glu Gly Val Pro Cys Val Leu Gln Gly Ala Pro Glu 8590 95 His Leu Pro His Leu His Gly Gly Ala Pro Arg Gly Leu His His Gln100 105 110 Pro Leu Gly Asp Ser Val Leu Pro Gln Gly Val Ser Arg Arg HisCys 115 120 125 Leu Gly Leu Gly Arg Leu Leu Pro Pro Ala Gly Gly Pro GluTrp Leu 130 135 140 Trp Trp Pro Pro Gly Gly Gly Ile Pro Gly Gln Gln ProHis Ala Gly 145 150 155 160 Ala Ala Glu Ala Gly Ile Asp Cys Thr Glu GlyPro Leu Ser Thr Thr 165 170 175 Leu Cys Gly Gln Pro Leu Ser Gln His LeuGlu Pro Tyr Ala His Thr 180 185 190 Gln Pro Ala Leu Pro Pro Gly Gly ThrAla Gly Ala Ser Leu Glu Pro 195 200 205 Ala Gly Arg Arg Gln Ala Ala AlaVal Arg Gly Gln Cys Gly Pro Leu 210 215 220 Arg Ala Ala Lys Ala Glu GlnVal Gly Arg Ala Ser Val Asp Arg Pro 225 230 235 240 Val Gln Pro Ala AlaLys Pro Gly Leu Pro His Lys Phe Cys Gln Gly 245 250 255 Thr Ala Thr GlnAla His Arg His Thr Ala Gly Pro Ala Ala Gln Pro 260 265 270 Gly Pro LeuAla Glu Phe Pro Gly Pro Ser Asp Pro Asn Asn Pro Ala 275 280 285 Ser GluGly Pro Gly Val Gly Thr Asp Pro Arg Asn Leu Gly Ala Ala 290 295 300 GlyArg Gln Val Ala Gly Asn Cys Gln Ala Pro Ala Pro Ala Cys Val 305 310 315320 Gln Pro Leu Arg Ala Gly Pro Val Ala Ala Gly Ala Lys Val Asp Leu 325330 335 Gln Ala Gly Cys Ala Arg Gly Ser Gly Ser Arg Ala Ala Leu Gln Cys340 345 350 Arg Gly Phe Ala Leu Leu Thr Met Pro Glu 355 360

We claim:
 1. An isolated polynucleotide encoding a polypeptidecomprising an amino acid sequence of SEQ ID NO:2.
 2. The polynucleotideof claim 1, wherein said polynucleotide comprises the nucleic acidsequence of SEQ ID NO:
 1. 3. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2.
 4. A nucleic acid of 15 to about 100base pairs comprising from 15 contiguous base pairs of SEQ ID NO: 1, orthe complement thereof.
 5. The nucleic acid of claim 4, comprising fromabout 20 contiguous base pairs of SEQ ID NO: 1, or the complementthereof.
 6. The nucleic acid of claim 4, comprising from about 25contiguous base pairs of SEQ ID NO:1, or the complement thereof.
 7. Thenucleic acid of claim 4, comprising from about 30 contiguous base pairsof SEQ ID NO: 1, or the complement thereof.
 8. The nucleic acid of claim4, comprising from about 40 contiguous base pairs of SEQ ID NO:1, or thecomplement thereof.
 9. The nucleic acid of claim 4, comprising fromabout 50 contiguous base pairs of SEQ ID NO: 1, or the complementthereof.
 10. The nucleic acid of claim 4, comprising from about 100contiguous base pairs of SEQ ID NO: 1, or the complement thereof.
 11. Anisolated peptide having between 10 and about 50 consecutive residues ofSEQ ID NO:2.
 12. The peptide of claim 11, comprising 15 consecutiveresidues of SEQ ID NO:2.
 13. The peptide of claim 11, comprising 20consecutive residues of SEQ ID NO:2.
 14. The peptide of claim 11,comprising 25 consecutive residues of SEQ ID NO:2.
 15. The peptide ofclaim 11, comprising 30 consecutive residues of SEQ ID NO:2.
 16. Thepeptide of claim 11, comprising 50 consecutive residues of SEQ ID NO:2.17. An expression cassette comprising a polynucleoticle encoding apolypeptide having the sequence of SEQ ID NO:2, wherein saidpolynucleotide is under the control of a promoter operable in eukaryoticcells.
 18. The expression cassette of claim 17, wherein said promoter isheterologous to the coding sequence.
 19. The expression cassette ofclaim 18, wherein said promoter is a tissue specific promoter.
 20. Theexpression cassette of claim 18, wherein said promoter is an induciblepromoter.
 21. The expression cassette of claim 18, wherein saidexpression cassette is contained in a viral vector.
 22. The expressioncassette of claim 21, wherein said viral vector is selected from thegroup consisting of a retroviral vector, an adenoviral vector, andadeno-associated viral vector, a vaccinia viral vector, and aherpesviral vector.
 23. The expression cassette of claim 17, whereinsaid expression cassette further comprises a polyadenylation signal. 24.A cell comprising an expression cassette comprising a polynucleotideencoding a polypeptide having the sequence of SEQ ID NO:2, wherein saidpolynucleotide is under the control of a promoter operable in eukaryoticcells, said promoter being heterologous to said polynucleotide.
 25. Amonoclonal antibody that binds immunologically to a polypeptidecomprising SEQ ID NO:2, or an immunologic fragment thereof.
 26. Themonoclonal antibody of claim 25, wherein the antibody further comprisesa detectable label.
 27. The monoclonal antibody of claim 26, wherein thelabel is selected from the group consisting of a fluorescent label, achemiluminescent label, a radiolabel and an enzyme.
 28. A hybridoma cellthat produces a monoclonal antibody that binds immunologically to apolypeptide comprising SEQ ID NO:2, or an immunologic fragment thereof.29. A polyclonal antisera, antibodies of which bind immunologically to apolypeptide comprising SEQ ID NO:2, or an immunologic fragment thereof.30. A isolated and purified nucleic acid that hybridizes, under highstringency conditions, to a DNA segment comprising SEQ ID NO: 1, or thecomplement thereof.
 31. The nucleic acid of claim 30, wherein saidnucleic acid is about 15 bases in length.
 32. The nucleic acid of claim30, wherein said nucleic acid is about 17 bases in length.
 33. Thenucleic acid of claim 30, wherein said nucleic acid is about 20 bases inlength.
 34. The nucleic acid of claim 30, wherein said nucleic acid isabout 25 bases in length.
 35. A method for constructing a recombinantadenovirus comprising: (a) providing a shuttle vector, said shuttlevector comprising an adenoviral inverted terminal repeat (ITR) sequence,an expression cassette comprising a promoter and a poly-A sequence, atransgene under the control of said promoter, and unique restrictionsites at the 5′- and 3′-ends of the ITR-promoter-transgene-poly-Asegment; (b) cutting at said restriction enzyme sites; (c) ligating thereleased segment into an adenoviral vector lacking the entire E1 and E3regions and transforming the resulting vector a bacterial host cell; (d)obtaining vector from said bacterial host cell and digesting the vectorto release the E1/E3-deleted adenovirus genome; and (e) transfecting theadenovirus genome into E1-expressing host cells.
 36. The method of claim35, wherein said transgene is Gene 26 (CACNA2D2), PL6, Beta* (BLU),Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21(NPRL2), or SEM A3Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.37. The method of claim 35, wherein said promoter is a cytomegalovirus(CMV) promoter and said poly A sequence is bovine growth hormone (BGH)poly A sequence.
 38. A method for constructing a recombinant adenoviruscomprising: (a) providing a shuttle vector comprising an adenoviralinverted terminal repeat (ITR) sequence, an expression cassettecomprising a promoter and poly-A signal sequence, a transgene under thecontrol of said promoter, a tetracycline resistance-off responsiveelement, and unique restriction sites at the 5′ and 3′ ends of theIRT-promoter-transgene-poly-A segment; (b) cutting at said restrictionenzyme sites; (c) ligating the released segment into an adenoviralvector comprising a tetracyclin resistant-off transactivator gene andlacking the entire E1 and E3 regions, and transforming the resultingvector a bacterial host cell; (d) obtaining vector from said bacterialhost cell and digesting the vector to release the E1/E3-deletedadenovirus genome; and (e) transfecting the adenovirus genome intoE1-expressing host cells.
 39. The method of claim 38, wherein saidtransgene is Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1),Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEMA3Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.
 40. The methodof claim 38, wherein said promoter is a cytomegalovirus (CMV) promoterand said poly A sequence is bovine growth hormone (BGH) poly A sequence.41. A shuttle vector comprising an adenoviral inverted terminal repeat(ITR) sequence, an expression cassette comprising a promoter and. poly-Asequence, a TetR-Off responsive element, and unique restriction sites atthe 5′- and 3′-ends of the ITR-promoter-poly-A segment.
 42. The shuttlevector of claim 41, wherein said promoter is a cytomegalovirus (CMV)promoter and said poly A sequence is bovine growth hormone (BGH) poly Asequence.
 43. The shuttle vector of claim 41, further comprising amultipurpose cloning site in said segment, positioned between saidpromoter and said poly-A sequence.
 44. An adenoviral vector comprising atetracycline resistant-off transactivator gene and lacking the entire E1and E3-regions.
 45. A method of diagnosing cancer in a subjectcomprising the steps of: (i) obtaining a biological sample from saidsubject; and (ii) assessing the expression of a functional Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3Gene 26 (CACNA2D2),PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1,101F6, Gene 21 (NPRL2), or SEM A3 product in sample.
 46. The method ofclaim 45, wherein said sample is a tissue sample.
 47. The method ofclaim 46, wherein said tissue sample is selected from the groupconsisting of brain, lung, liver, spleen, kidney, lymph node, smallintestine, blood cells, pancreas, colon, stomach, cervix, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,oral tissue, bone marrow and blood tissue.
 48. The method of claim 45,wherein said assessing comprises detecting a nucleic acid encoding Gene26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3Gene 26 (CACNA2D2),PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1,101F6, Gene 21 (NPRL2), or SEM A3.
 49. The method of claim 48, whereindetecting comprises amplification said nucleic acid.
 50. The method ofclaim 48, wherein detecting comprises nucleic acid hybridization. 51.The method of claim 48, wherein detecting comprises sequencing.
 52. Themethod of claim 45, wherein said assessing comprises detecting a Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3Gene 26 (CACNA2D2),PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1,101F6, Gene 21 (NPRL2), or SEM A3 polypeptide.
 53. The method of claim52, further comprising ELISA.
 54. The method of claim 52, furthercomprising immunohistochemistry.
 55. The method of claim 45, whereinsaid assessing comprises wild-type or mutant oligonucleotidehybridization, and said oligonucleotide configured in an array on a chipor wafer.
 56. The method of claim 45, further comprising the step ofcomparing the expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3 with the expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU),Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21(NPRL2), or SEM A3 in normal samples.
 57. The method of claim 56,wherein the comparison involves evaluating the level of Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 expression.
 58. Anon-human transgenic animal lacking at least one functional Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 allele.
 59. Thenon-human transgenic animal of claim 58, wherein said animal lacks bothfunctional alleles of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3.
 60. A non-human transgenic animal that overexpresses Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 as compared to asimilar non-transgenic animal.
 62. A non-human transgenic animal, thegenome of which comprises an expression cassette comprising a Gene 26(CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2(RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 under the control ofan inducible promoter.
 63. A method for suppressing growth of a tumorcell comprising contacting said cell with an expression cassettecomprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta*(BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene21 (NPRL2), or SEM A3; and (b) a promoter active in said tumor cell,under conditions permitting the uptake of said nuclcic acid by saidtumor cell.
 64. The method of claim 63, wherein said tumor cell isderived from a tumor a selected from the group consisting of braintumor, lung tumor, liver tumor, spleen tumor, kidney tumor, lymph nodetumor, small intestine tumor, blood cell tumor, pancreatic tumor, colontumor, stomach tumor, cervix tumor, breast tumor, endometrial tumor,prostate tumor, testicle tumor, ovarian tumor, skin tumor, head and necktumor, esophageal tumor, oral tissue tumor, and bone marrow tumor. 65.The method of claim 63, wherein said nucleic acid is contained in aviral vector.
 66. The method of claim 65, wherein said viral vector is aretroviral vector, an adenoviral vector, an adeno-associated viralvector, a vaccinia viral vector, or a herpesviral vector.
 67. The methodof claim 66, wherein said viral vector is an adenoviral vector.
 68. Themethod of claim 63, wherein said nucleic acid is contained in aliposome.
 69. A method of altering the phenotype of a tumor cellcomprising contacting said cell with an expression cassette comprising:(a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3; and (b) a promoter active in said tumor cell, underconditions permitting the uptake of said nucleic acid by said tumorcell.
 70. The method of claim 69, wherein the phenotype is selected fromthe group consisting of proliferation, migration, contact inhibition,soft agar growth, cell cycling, invasiveness, tumorigenesis, andmetastatic potential.
 71. The method of claim 69, wherein said promoteris a cytomegalovirus (CMV) promoter.
 72. A method of inhibiting cancerin a subject suffering therefrom comprising administering to saidsubject an expression cassette comprising: (a) a nucleic acid encodingGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide; and(b) a promoter active in tumor cells of said subject, whereby expressionof said polypeptide inhibits said cancer.
 73. The method of claim 72,wherein said subject is a human.
 74. The method of claim 72, whereinsaid nucleic acid encodes Gene 26 (CACNA2D2).
 75. The method of claim72, wherein said nucleic acid encodes PL6.
 76. The method of claim 72,wherein said nucleic acid encodes Beta* (BLU).
 77. The method of claim72, wherein said nucleic acid encodes LUCA-1 (HYAL1).
 78. The method ofclaim 72, wherein said nucleic acid encodes LUCA-2 (HYAL2).
 79. Themethod of claim 72, wherein said nucleic acid encodes 123F2 (RASSF1).80. The method of claim 72, wherein said nucleic acid encodes Fus1. 81.The method of claim 72, wherein said nucleic acid encodes 101F6.
 82. Themethod of claim 72, wherein said nucleic acid encodes Gene 21 (NPRL2).83. The method of claim 72, wherein said nucleic acid encodes SEM A3.84. The method of claim 72, wherein said cancer is a selected from thegroup consisting of brain cancer, lung cancer, liver cancer, spleencancer, kidney cancer, lymph node cancer, small intestine cancer, bloodcell cancer, pancreatic cancer, colon cancer, stomach cancer, cervixcancer, breast cancer, endometrial cancer, prostate cancer, testiclecancer, ovarian cancer, skin cancer, head and neck cancer, esophagealcancer, oral tissue cancer, and bone marrow cancer.
 85. The method ofclaim 72, wherein said expression cassette is contained in a viralvector.
 86. The method of claim 85, wherein said viral vector is aretroviral vector, an adenoviral vector, an adeno-associated viralvector, a vaccinia viral vector, or a herpesviral vector.
 87. The methodof claim 86, wherein said viral vector is an adenoviral vector.
 88. Themethod of claim 72, wherein said expression cassette is contained in alipsome.
 89. The method of claim 72, wherein said expression cassettefurther comprises a poly-A sequence.
 90. The method of claim 89, whereinsaid poly-A sequence is bovine growth hormone (BGH) poly-A sequence. 91.The method of claim 72, wherein said expression cassette is administeredintratumorally, in the tumor vasculature, local to the tumor, regionalto the tumor, or systemically.
 92. The method of claim 72, furthercomprising administering a chemotherapuetic agent to said subject. 93.The method of claim 92, wherein said chemotherapeutic comprisescisplatin (CDDP), carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulian, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabien,navelbine, famesyl-protein tansferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate.
 94. The methodof claim 72, further comprising administering radiation to said subject.95. The method of claim 94, wherein said radiation is delivered local toa cancer site.
 96. The method of claim 94, wherein said radiation iswhole body radiation.
 97. The method of claim 94, wherein said radiationcomprises γ-rays, X-rays, accelerated protons, microwave radiation, UVradiation or the directed delivery of radioisotopes to tumor cells. 98.The method of claim 72, further comprising administering a secondanticancer gene to said subject.
 99. The method of claim 98, whereinsaid second anticancer gene is a tumor suppressor.
 100. The method ofclaim 98, wherein said second anticancer gene is an inhibitor ofapoptosis.
 101. The method of claim 98, wherein said second anticancergene is an oncogene antisense construct.
 102. A method of treating asubject with cancer, comprising the step of administering to saidsubject a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2(HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3polypeptide.
 103. The method of claim 102, wherein said cancer is aselected from the group consisting of brain cancer, lung cancer, livercancer, spleen cancer, kidney cancer, lymph node cancer, small intestinecancer, blood cell cancer, pancreatic cancer, colon cancer, stomachcancer, cervix cancer, breast cancer, enclometrial cancer, prostatecancer, testicle cancer, ovarian cancer, skin cancer, head and neckcancer, esophageal cancer, oral tissue cancer, and bone marrow cancer.104. The method of claim 102, wherein said polypeptide is containedwithin a liposome.
 105. The method of claim 104, wherein said liposomeis comprised of N-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium(DOTAP) and cholesterol.
 106. The method of claim 102, wherein thesubject is human.
 107. A method of screening a candidate substance foranti-tumor activity comprising the steps of: (i) providing a celllacking a functional Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1(HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2),or SEM A3 polypeptide; (ii) contacting said cell with said candidatesubstance; and (iii) determining the effect of said candidate substanceon said cell.
 108. The method of claim 107, wherein said cell is a tumorcell.
 109. The method of claim 107, wherein said determining comprisescomparing one or more characteristics of the cell in the presence ofsaid candidate substance with the same one or more characteristics of asimilar cell in the absence of said candidate substance.
 110. The methodof claim 109, wherein said characteristic is Gene 26 (CACNA2D2), PL6,Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1,101F6, Gene 21 (NPRL2), or SEM A3 expression, phosphatase activity,proliferation, metastasis, contact inhibition, soft agar growth, cellcycle regulation, tumor formation, tumor progression, metastasis andtissue invasion.
 111. The method of claim 107, wherein said candidatesubstance is a chemotherapeutic or radiotherapeutic agent.
 112. Themethod of claim 107, wherein said candidate substance is selected from asmall molecule library.
 113. The method of claim 107, wherein said cellis contacted in vitro.
 114. The method of claim 107, wherein said cellin contacted in vivo.
 115. A method of screening a candidate substancefor anti-tumor activity comprising the steps of: (i) providing a cell;(ii) contacting said cell with said candidate substance; and (iii)determining the effect of said candidate substance on expression of aGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide.116. A method of producing a Beta* polypeptide in a host cellcomprising: (a) providing an expression cassette comprising a nucleicacid encoding Beta* operably linked to an promoter active in said hostcell; (b) transferring said expression cassette into said host cell; and(c) culturing said host cell under conditions permitting expression ofsaid Beta* polypeptide.
 117. A method of diagnosing cancer in a subjectcomprising the steps of: (i) obtaining a biological sample from saidsubject; and (ii) detecting hypermethylation of the promoter region ofGene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2),123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.